Effects And Mechanisms Of Lung Cancer Derived Exosomes On The Remodeling Of Commensal Bacteria

Background: Tumor microenvironment(TME)is a dynamic niche for tumorigenesis and progression.Emerging evidence has shed light on the unusual role of commensal microbiota in tumor microenvironment across various tumors.Microbiota can have long-time exist in the TME or even in tumor cells without uncontrolled proliferation and fatal infection to tumor cells,and it can interact with tumor cells when they coexist.Specific microbiota can contribute to carcinogenesis by inducing tumor-associated inflammation,producing immunosuppressive factors,and releasing detrimental metabolites.But less is known about how the tumor cells can exert effects and modulate the commensal microbiota.Its potential mechanism remains to be deeply studied.Objective: To observe the effects of exosomes from lung adenocarcinoma on the structure and function of the lung commensal microbiota,and to reveal the potential molecular mechanism.Methods: 56 lung specimens from clinical operation were collected in this study(including32 adjacent tissues and 23 tumor tissues).16 S r RNA sequencing was performed to detect the enriched bacteria in lung adenocarcinoma.Two lung cancer cell lines A549,H1299 and human bronchial epithelial cell line BEAS-2B were cultured with the exosome-free medium,the culture supernatants were collected,and exosomes were extracted by ultracentrifugation.The morphology of exosomes was observed by transmission electron microscope,the diameter distribution of exosomes was analyzed by nanoparticle tracking Analysis(NTA),the expression of markers for exosomes was detected by Western blotting.Standard strain of enriched bacteria was expanded and co-incubated with fluorescent-labeled exosomes to observe the internalization of exosomes.After the treatment of exosomes,live & dead staining was used to evaluate the viability of bacteria,and growth curve was drawn.The effects of exosomes from different cell lines on bacterial morphology were observed by Gram staining,HCC-Amino-D-alanine tracing,transmission electron microscope and scanning electron microscope.The peptidoglycan and teichoic acid contents were detected to evaluate the cell wall synthesis.Then,crystal violet staining,Giemsa staining and bacterial coating plate experiment were used to evaluate the effects of exosomes from different cell sources on bacterial biofilm formation and adhesion to host cells.Hemolysis test,Western blotting and cell culture experiment were applied to detect the virulence change of the bacterial,and the RNA was extracted for transcriptome sequencing and q PCR verification to explore the potential molecular mechanism under the biological phenomena.Results: Staphylococcus aureus(S.aureus)was enriched in the adenocarcinoma tissues compared with the adjacent tissues.the NTA assay showed the size range of all exosomes were 30-150 nm in diameter with discoid in appearance.The Western blotting showed that exosomes can express CD9,CD63,and TSG101 as transmembrane markers.The confocal imaging results indicated that different exosomes can be taken up by S.aureus.Lung cancer-derived exosomes can reduce the peptidoglycan content of S.aureus.The exosomes derived from A549 and H1299 can restrain the biofilm formation,adhesive capacity,and bacterial virulence.Transcriptome sequencing and q PCR verification showed that the expression of Arl S-Arl R two-component system and Mgr A were significantly up-regulated in A549 and H1299 exosome treatment groups.Besides,ebh(a kind of extracellular matrix binding protein)and ebps(elastin-binding protein of S.aureus)which were associated to adhesion and biofilm formation were down-regulated significantly,the virulence coding gene hla was down-regulated in S.aureus after A549 and H1299-drived exosomes treatment.Conclusion: In the present study,we found that the tumor tissues of patients with lung adenocarcinoma were enriched with S.aureus relative to the adjacent tissues,and the lung cancer-derived exosomes can be internalized by S.aureus,the treatment of lung cancer-derived exosomes altered the bacterial morphology,reduced the adhesive ability,biofilm formation and the virulence of S.aureus,the activation of Arl S-Arl R two-component system may be one of the potential mechanisms under the above phenomenon.It is a novel complement of the crosstalk between tumor cells and the commensal microbiota.