The Investigation On The Role Of Cancer-associated Fibroblasts In Metastasis And Chemoresistance Of NSCLC Cells And Underling Mechanisms

ObjectiveLung cancer is one of the most common malignancies,and its incidence of morbidity and mortality are also the highest in the world.According to the histopathological features,lung cancer could be divided into two major categories:small cell lung cancer?SCLC?and non-small cell lung cancer?NSCLC?,of which85%is NSCLC.At present,the most effective threapeutics for lung cancer is surgical treatment.However,it commonly could not achieve radical cure due to the difficulty in early detection or distant metastasis.The 5-year survival rate of NSCLC patients is less than 20%.Chemotherapy is another important therapy method for NSCLC.However,inevitable acquired resistance and metastasis always lead to the failure of treatment.The invasion and metastasis of tumor cells is one major cause of the failure of cancer treatments.Drud resistance of tumor cells is another obstacle to complete clear tumors.Therefore,investigations about the underling mechanisms of metastasis and drug resistance in cancer cells could be urgent and meaningful to improve the therapeutic effect of patients with cancer.Besides the mechanisms in cancer cells,the invasion and metastasis of cancer cells also could be affected by tumor microenvironment.Researches have shown the complex interactions between cancer cells and microenvironment:cancer cells can affect the proliferation and distribution of cells in tumor microenvironment by secreting a variety of cytokines;at the same time,the latter could also respond,such as providing skeleton support for tumor tissue,secreting various factors to promote angiogenesis,promoting the growth,invasion and chemoresistance of cancer cells.Cancer-associated fibroblasts?CAFs?,the fibroblasts in tumor tissue are the major cell components of the tumor microenvironment.By secreting various cytokines to promote tumor angiogenesis and induce the epithelial-mesenchymal transition?EMT?of tumor cells,CAFs could break down the steady-state between tissues and make a suitable microenvironment for tumor.A large number of studies have shown that CAFs can promote the proliferation,invasion,metastasis and chemoresistance of various cancers such as breast cancer,liver cancer,gastric cancer,colorectal cancer,and ovarian cancer.This study mainly focus on the effects and the mechanisms of CAFs on the proliferation,invasion,metastasis and chemoresistance of NSCLC cells,to provide new ideas for the treatment of lung cancer.Contents and Methods1.Fibroblast cell lines isolation and identifications:CAFs and normal fibroblasts?NFs?were isolated from clinical lung cancer surgical specimens by tissue grinding and digestion.After weeks'culture,further identifications were conducted by microscopic observation of morphology,PCR,immunofluorescence and western blot detection of?-SMA,FAP and other fibroblast marker proteins.2.The effects of CAFs on the proliferation,invasion and metastasis of NSCLC cells:Microscopic observation,wound healing,transwell and CCK8 assays were conducted to analyze the effects of cancer-associated fibroblast conditioned medium?CAF-CM?on the morphology,invasion,migration,and proliferation of NSCLC cell lines,normal fibroblast conditioned medium?NF-CM?as a negative control.The transcription and expression levels of invasion and metastasis related genes:MMP2,MMP9,VEGF and EMT-related molecules:E-cadherin,N-cadherin and Vimentin were detected by Western blot and PCR.3.The mechanism of CAFs promoting the proliferation,invasion and metastasis of NSCLC cells:ELISA was conducted to detect the levels of IL-6 in CAF-CM and NF-CM.PCR and Western blot were used to detect the transcription and expression levels of IL-6 in NSCLC cell lines stimulated by CAF-CM or NF-CM.Recombinant IL-6 and IL-6 neutralizing antibodies were also used to clarify the role of IL-6 in the supernatant.PCR and Western blot to analyse the transcription and expression levels of IL-6 downstream moleculars including p-JAK2 and p-STAT3 in NSCLC cell lines stimulated by CAF-CM and NF-CM.JAK2 inhibitor AG490 and STAT3 inhibitor Stattic were used to identify the role of the JAK2/STAT3 pathway.4.The effects of CAFs on proliferation,invasion and metastasis of NSCLC cells in vivo:The nude mice xenograft model was established to observe the effect of CAFs on the biological behavior of NSCLC cells in vivo.NSCLC cell line A549mixed with CAFs or alone were injected subcutaneously into BALB/c nude mice.The sizes of tumors were monitored and the tumor tissues were dissected after 6weeks.Western blot was used to detect theexpression levels of related genes in tumor tissue.5.The effects of CAFs on the cisplatin sensitivity of NSCLC cells:CCK8 assay was conducted to observe the changes in IC₅₀ of cisplatin to NSCLC cell lines A549and H661 stimulated by CAF-CM.6.The molecular mechanisms of CAFs promoting the cisplatin resistance of NSCLC cells:Western blot and PCR were used to detect the transcription and expression levels of ANXA3 in CAF-CM stimulated NSCLC cell lines.CCK8 assay and Western blot were conducted to detect the changes of cisplatin sensitivity and the levels of apoptosis-related proteins:p-JNK,Survivin,cleaved Caspase8 and cleaved Caspase3 in ANXA3 overexpression or knockdown NSCLC cells stimulated by CAF-CM.7.The effects of CAFs on cisplatin sensitivity of NSCLC cells in vivo:The NSCLC cell line A549 mixed with CAFs or alone were injected subcutaneously into BALB/c nude mice,and cisplatin was injected intraperitoneally after tumor formation.After 6 weeks,the tumor tissues were dissected and the levels of proteins in tumor tissues were detected by Western blot.Results1.The shapes of fibroblasts extracted from clinical tissues were all spindle,and the transcription and expression levels of fibroblast markers including?-SMA and FAP were all highl.All of these indicated that the isolated fibroblasts were cancer-associated fibroblasts.2.Compared with related NFs,CAFs could significantly promote the invasion,metastasis and proliferation capacities,upregulate the expression of metastasis-associated genes MMP2,MMP9,VEGF,and promote the epithelial-mesenchymal transition?EMT?of NSCLC cells.The level of IL-6 was higher in CAF-CM than NF-CM.IL-6 neutralizing antibody could significantly weaken the promotion ability of CAF-CM to the invasion and metastasis of NSCLC cells.Normal culture media with recombinant human IL-6 could cause similar effects with CAF-CM.The level of p-JAK2 and p-STAT3 were both upregulated in NSCLC cells treated with CAF-CM or recombinant human IL-6.JAK2 inhibitor:AG490 and STAT3 inhibitor:Stattic could undermine these promotion effects of CAF-CM and recombinant human IL-6.The result of nude mice xenograft model experiment showed that:Mix CAFs with A549 could significantly promote tumor growth.The expression level changes of related genes in tumor tissues were consistent with in vitro fingings.3.CAF-CM could increase sensitivity of NSCLC cells to cisplatin.The expression level of ANXA3 in CAFs was significantly higher than NFs,and the expression of ANXA3 in NSCLC cells was up-regulated after stimulated by CAF-CM.In the NSCLC cells,overexpress ANXA3 or stimulated by CAF-CM,the levels of p-JNK and Survivin were increased,while cleaved Caspase8 and cleaved Caspase3were decreased.Knocked down ANXA3 in NSCLC cells by siRNA,the levels of p-JNK and Survivin were decreased,and the levels of cleaved Capase8 and cleaved Caspase3 were increased.After inhibiting the JNK pathway,the effects of CAF-CM and ANXA3 on the cisplatin resistance of NSCLC cells were significantly impaired.Animal experiments also showed that the tumor volumes of the mixed group with CAFs was significantly greater than those of the single A549 group with cisplatin treament,and the related protein level changes in tumor samples were consistent with in vitro findings.ConclusionsWe successfully isolated CAFs from clinical lung cancer tissues.We investigated the effects of CAFs on NSCLC cells,and found that CAFs can promote the invasion and metastasis capacities of NSCLC cells by secreting IL-6.By activating JAK2/STAT3 pathway in NSCLC cells,IL-6 could further promote EMT,and up-regulate the expression of invasive and metastasis-associated genes.We also studied the roles of CAFs in chemoresistance,and found that:by up-regulating the expression of ANXA3 in NSCLC cells,CAFs could activate JNK/Survivin pathway and promote the cisplatin resistance of NSCLC cells.