Exosomes From Dormant Lung Adenocarcinoma Cells Activate Cancer-associated Fibroblasts

Objective: Investigate the mechanism by which exosomes derived from dormant lung adenocarcinoma cells activate cancer associated fibroblasts to reconstruct the extracellular matrix,providing a novel idea for decoding the mechanism of tumor dormancy.Methods:(1)High-dose cisplatin was used to construct the dormant lung adenocarcinoma cells base on A549 cells.(2)Exosomes were extracted from the culture supernatant of normal and dormant A549 cells by precipitation method.(3)A549-luciferase cells were subcutaneously inoculated into nude mice to construct tumor-bearing nude mouse models.Exosomes were injected into the subcutaneous tumor.Tumor metastasis in each group was assessed by in vivo imaging system.(4)The markers of cancer-associated fibroblasts were detected by western blot;the expression of matrix related genes was detected by Q-PCR.Transwell assay and in vitro matrix model were used to evaluate the invasion ability and collagen contraction ability of fibroblasts incubated with exosomes.(5)The key gene was screened by RNA-seq;the effect of key gene expression level on the invasion ability and collagen contraction ability of fibroblasts was evaluated by Transwell assay and in vitro matrix model.(6)Exosome proteomics were analyzed to screen and verify the involvement of the essential proteins in activating fibroblasts.(7)Survival analysis was performed using GEPIA database.Results:(1)Exosomes derived from A549 cells and dormant A549 cells both promoted lung cancer metastasis.(2)After incubating the exosomes from dormant A549 cells,the expression of α-SMA in fibroblasts was upregulated.The m RNA expression of TGFB1,COL1A1 and COL3A1 was upregulated,while COL4A1 were downregulated.The capacity for contracting collagen fibers and invasion was enhanced in fibroblasts.(3)KLF10 knockdown inhibited the contractility and invasion abilities of the fibroblasts activated by exosomes from dormant A549 cells.KLF10 overexpression activated related abilities in fibroblasts.(4)KLF10activated TGF-β signaling by phosphorylation of Smad2 with positive feedback,which induced the activation of cancer associated fibroblasts.(5)The exosomal ITGB6 from dormant A549 cells activated TGF-β pathway and KLF10 positive feedback loop.Overexpression of ITGB6 enhanced the invasion of fibroblasts.(6)High ITGB6 expression was associated with worse disease-free survival times in the patients with non-small cell lung cancer.Conclusions:(1)Exosomes from lung adenocarcinoma cells and dormant lung adenocarcinoma cells both promote lung cancer metastasis.(2)Exosome ITGB6 from dormant lung adenocarcinoma cells activates TGF-β pathway and KLF10 positive feedback loop,which induces the activation of cancer associated fibroblasts.(3)ITGB6 may be a potential biomarker for predicting postoperative recurrence and metastasis in patients with non-small cell lung cancer.