| Objectives:As a first-line treatment modality for non-small cell lung cancer(NSCLC),epidermal growth factor receptor-tyrosine kinase inhibitor(EGFR-TKI)has achieved remarkable progression in improving patient survival,but its inevitable problem of acquired resistance greatly limits the long-term prognosis of patients.The occurrence of acquired resistance not only depends on the characteristics of tumor cells but is also associated with the tumor microenvironment.As a major component of the tumor microenvironment,cancer-associated fibroblasts(CAF)play an important role in promoting the occurrence of acquired resistance.Our study found that G protein coupled estrogen receptor 1(GPER1)was highly expressed in cancer-associated fibroblasts compared with normal fibroblasts(NF).However,whether CAF with high GPER1 are involved in the occurrence of osimertinib resistance and the underlying mechanism remain unclear.Methods:(1)Peritumoral normal lung tissue and tumor tissue from non-small cell lung cancer patients with EGFR activating mutation were collected and enzymatically digested to extract NF and CAF,respectively.Expression of α-SMA and FAP were assessed by Western blot and cellular immunofluorescence to identify CAF(2)Differential GPER1 expression in NF and CAF was assessed by Western blot,cellular immunofluorescence and tissue immunofluorescence(3)CCK8 assays,cellular colony formation assays and Edu cell proliferation assays were performed to test whether CAF with high expression of GPER1 could induce the emergence of acquired resistance to osimertinib in NSCLC,and whether knockdown of its protein expression of GPER1 could reverse the acquired resistance.(4)Western blot and ELISA assays were performed to detect the expression of TGF-β1,TGF-β2 and TGF-β3 in NF and CAF as well as the changes of TGF-β1,TGF-β2 and TGF-β3 after knocking down GPER1.(5)Immunohistochemistry staining assays were performed to detect the expression levels of GPER1 and TGF-β2 and their correlation in CAF.(6)Cellular colony formation assays and Edu cell proliferation assays were performed to test whether CAF overexpressing GPER1 could induce the occurrence of osimertinib resistance through TGF-β2.(7)Mouse subcutaneous tumor xenograft model was constructed to verify the role of CAF overexpressing GPER1 in inducing the emergence of osimertinib resistance and whether knocking down GPER1 could reverse the acquired resistance.Results : Western blot,cellular immunofluorescence,and tissue immunofluorescence experiments revealed that compared to NF,CAF highly expressed their markers α-SMA and FAP,while GPER1 in CAF also showed the high expression status.Stable knockdown of GPER1 in CAF was constructed by using lentivirus.We found that CAF could induce the emergence of osimertinib resistance by CCK8 assays,cellular colony formation assays and Edu cell proliferation assays,and their effect of promoting the emergence of drug resistance was reversed after knocking down GPER1.CAF were found to overexpress TGF-β1 and TGF-β2 by Western blot,and knockdown of GPER1 downregulated TGF-β2expression and secretion in CAF,without affecting TGF-β1 and TGF-β3 expression.The results of Western blot assay revealed that CAF overexpressing GPER1 induced the emergence of osimertinib resistance in NSCLC by promoting epithelial mesenchymal transition(EMT)via TGF-β2.In vivo,CAF induced the emergence of osimertinib resistance in NSCLC,and knockdown of GPER1 reversed acquired resistance.Conclusions:This study showed for the first time that CAF expressed high levels of GPER1 compared to NF in NSCLC.CAF with high expression of GPER1 promoted NSCLC to undergo epithelial mesenchymal transition by secreting TGF-β 2,which in turn induced osimertinib resistance.Knockdown of GPER1 in CAF could inhibit the secretion of TGF-β2 to reverse acquired resistance.Thus,GPER1 in CAF is a potential therapeutic target for reversing osimertinib resistance in non-small cell lung cancer. |
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