The Molecular Mechanism Of Lung Cancer Cell-derived Exosomes Regulating Intrapulmonary Metastasis Of Lung Cancer

BackgroundLung cancer is one of the most common malignant tumors with the highest morbidity and mortality rate.More than 70%of lung cancer patients’ death is caused by metastasis.Among them,intrapulmonary metastasis is one of the most common metastases of lung cancer.Recent studies have shown that tumor-derived exosomes could be transported to the new tissue through the blood circulation,mediating the communication between the primary tumor cells and the new metastatic site,which contribute to the establishment of pre-metastatic niche by modifying the local micro-environment of the metastatic site,thus providing suitable condition for the colonization of primary tumor cells.This is the widely accepted theory of "seed and soil".Exosomes are lipid bilayer-encapsulated microparticles(30-100 nm)formed in multivesicular bodies,which release exosomes into the extracellular milieu by fusing with cytomembrane.Exosomes could be produced by various types of cells and serve as mediators in intercellular communications by transporting information cargo,such as proteins,lipids,and nucleic acids.Previous studies have demonstrated that exosomes contain a large amount of miRNAs,and these exosomal miRNAs are shown to lead to tumor metastasis.The interaction between tumor cells and stromal cells in the tumor microenvironment plays an important role in tumor progression.Tumor associated fibroblasts,the main stromal cell component in various solid tumors,are believed to be involved in all stages of tumor progression,and it could secrete multiple factors to mediate tumor metastasis.It has been reported that tumor-derived exosomal miRNAs could promote tumor progression by regulating fibroblasts.However,the relevance between lung tumor-derived exosomal miRNAs and fibroblast mediated lung tumor metastasis is still unknown.Therefore,exploring the role of lung cancer-derived exosomal miRNAs on fibroblast-tumor cell interaction may provide new targets for tumor treatment.ObjectiveIn this study,we aim to investigate the mechanism of lung cancer cell-derived exosomal miRNAs in mediating intrapulmonary metastasis by regulating fibroblasts.Methods1.Isolate and identify exosomes from the culture supernatant of lung cancer cells using standard ultracentrifugation method.2.Cy5.5-labeled LLC-exosomes were injected into mice via tail vein,and lung tissues were collected to track exosomes by flow cytometry and immunohistochemistry.In vitro,Cy5.5-labeled LLC-exosomes were co-cultured with fibroblasts,and the whereabouts of the exosomes were observed by a confocal microscope.3.The role of LLC-exosomes on proliferation and migration of fibroblasts were observed by flow cytometry and Transwell assay.4.LLC-exosomes and LLC were injected into mice via tail vein to construct an animal model.Then we observed the effect of exosomes on lung cancer metastasis and immune cell population in the lung.5.The miRNA in exosomes and the prediction of miRNA target genes were detected by microarray analysis,and the effect of exosomes on genes in fibroblasts was detected by transcriptome sequencing.6.Reverse transcription polymerase chain reaction(qRT-PCR)and western blot(WB)were performed to observe the effects of LLC-exosomes on Nfkbid,inflammatory cytokines Il-6,Ccl1,Ccl2,Ccl5 and Cxcl2 and NF-κB signaling pathway in fibroblasts.7.The effects of exosomal miR-3473b on Nfkbid,inflammatory factors and NF-κB signaling pathway in fibroblasts were detected by qRT-PCR and WB.8.The effects of exosomal miR-3473b on lung metastasis and the number of B cells in lung were observed by injecting LLC-exosomes,miR-3473b mimic or inhibitor and LLC cells into mice through the tail vein.Results1.Flow cytometry analysis revealed that 54.1%of Cy5.5-labeled LLC-exosomes were absorbed by CD45-FSP(fibroblast specific protein)positive cells.Immunofluorescence results showed that LLC-exosomes were absorbed by alpha-smooth muscle actin+(α-SMA+)cells.2.The proliferation of fibroblasts was enhanced by the stimulation of LLC-exosomes.Whereas LLC-exosomes had no effect on the migration of fibroblasts.Besides,LLC-exosomes promoted lung cancer metastasis in mice and promoted the accumulation of B cells in the lung.3.LLC-exosomes activated the NF-κB signaling pathway and increased the expression of inflammatory factors such as Il-6,Ccl1,Ccl2,Ccl5 and Cxcl2 of fibroblasts by down-regulating the Nfkbid.LLC-exosomal miR-3473b plays an important role in this process.4.LLC-exosomal miR-3473b inhibitor significantly inhibits LLC-exosomes mediated intrapulmonary colonization of lung tumor cells.ConclusionLung cancer cell-derived exosomal miR-3473b activates its NF-κB signaling pathway and inflammatory factors expression by down-regulating the Nfkbid gene of fibroblasts,and ultimately promotes intrapulmonary colonization of lung tumor cells.Targeting miR-3473b can be an important target for preventing metastasis after lung cancer treatment.