The Role And Mechanism Of Cafs In Regulation Of Glucose Metabolic Reprogramming In NSCLC

Objective:We previously found that the gap junctional intercellular communication(GJIC),which was composed of Cx43,contributed to the invasiveness,migration,EMT of NSCLC.However,the role of GJIC in participating in the metablic coupling between CAFs and NSCLC remains unknown.We aim to investigate the role and mechanism of CAFs in regulation of metabolic reprogramming in NSCLC in this study.Methods:1.The primary CAFs and paired NFs were isolated from human lung cancer and adjacent normal tissues.Western blotting(WB)and immunofluorescence(IF)experiments were performed to confirmed characteristics and purity of primary fibroblasts.2.For direct coculture,fibroblasts were mixed with NSCLC cells in a 5:1 ratio and were seeded into T25 flasks.3.The level of glucose uptake and lactate production of monocultured and co-cultured CAFs were measured using glucose assay kit,lactate assay kit,respectively.Meanwhile,the key glycolytic enzymes,pyruvate kinase M2(PKM2)and lactate dehydrogenase isoform A(LDH-A)activity were detected by PKM2 and LDH-A enzyme activity assays.4.The level of lactate,citric acid,pyruvate,acetyl coenzyme A(acetyl-CoA),alpha-ketoglutaric acid(α-KG),adenosine triphosphate(ATP)of monocultured and co-cultured NSCLC cells were measured using glucose assay kit,lactate assay kit,citric acid assay kit,pyruvate assay kit,acetyl-CoA assay kit,α-KG assay kit,ATP assay kit,respectively.Meanwhile,lactate dehydrogenase isoform B(LDHB)activity were detected by LDH-B enzyme activity assay.5.The expressions of α-smooth muscle actin(α-SMA),Cx43,LDH-B in 82 NSCLC tissues and paired normal tissues were detected by immunohistochemistry(IHC).Results:1.CAFs displayed spindle-shaped,flattenedand fibroblast-like morphology similar to NFs.WB and immunofluorescence staining showed that CAFs strongly expressed fibroblast-specific biomarkers FAP-α and α-SMA,whereas NFs weakly expressed these two biomarkers(p<0.05).Moreover,both CAFs and NFs also expressed the mesenchymal marker vimentin but did not express the epithelial marker E-cadherin,while only the epithelial cancer cells A549 expressed the epithelial marker E-cadherin(p<0.05).2.The mixed CAFs and NSCLC cells grown to more than 90% confluency,suggesting that we successfully constructed a direct co-culture system of CAFs and NSCLC cells.3.Glucose uptake,lactate production,and activity of PKM-2 and LDH-A were significantly increased in CAFs cocultrued with NSCLC cells compared with CAFs cultured alone(p<0.05).4.Glucose uptake and lactate production were significantly decreased in NSCLC cells cocultrued with CAFs compared with NSCLC cells cultured alone(p<0.05).LDH-B activity,pyruvate,acetyl-CoA and TCA metabolites(citric acid,α-KG and ATP)levels were markedly increased in NSCLC cells in the presence of CAFs(p<0.05).5.Inhibition of GJIC by Cx43 knockdown in both CAFs and NSCLC cells reduced LDH-B activity as well as pyruvate,acetyl-CoA and TCA metabolites levels in cocultured NSCLC cells,whereas enhancement of GJIC by Cx43 overexpression in both CAFs and NSCLC cells elicited enhanced LDH-B activity as well as pyruvate,acetyl-CoA and TCA metabolites levels in cocultured NSCLC cells(p<0.05).Moreover,pretreatment of CAFs with glycolytic activator Fructose-1,6-bisphosphate(FBP)could not affect the reduced LDH-B activity as well as pyruvate,acetyl-CoA and TCA metabolites levels in GJIC-deficient cocultured NSCLC cells,while pretreatment of CAFs with glycolytic inhibitor 2-deoxy-dglucose(2-DG)could cause further enhanced lactate level and further reduced LDH-B activity as well as pyruvate,acetyl-CoA and TCA metabolites levels in these NSCLC cells(p<0.05).In addition,2-DG could partially antagonize the CAFs-induced decrease in lactate level and increase in LDH-B activity as well as pyruvate,acetyl-CoA and TCA metabolites levels in GJIC-enhanced cocultured NSCLC cells,while FBP could further strengthen the CAFs-induced decrease in lactate level and increase in LDH-B activity as well as pyruvate,acetyl-CoA and TCA metabolites levels in these NSCLC cells(p<0.05).6.The levels of α-SMA,Cx43 and LDH-B were significantly elevated in NSCLC samples compared to matched adjacent noncancerous tissues.Α-SMA was strongly expressed in stroma cells but not in cancer cells,whereas LDH-B was significantly expressed in cancer cells but not in tumor stroma.However,Cx43 was detected at the intercellularboundaries of cancer cells and tumor stroma.Moreover,accumulation of α-SMA and Cx43-positive CAFs were predominantly observed at the invasive front of NSCLC lesions.High expression of α-SMA,Cx43 or LDH-B was significantly correlated with TNM stage and NSCLC metastasis(p<0.05).Furthermore,high biexpression of α-SMA and Cx43,Cx43 and LDH-B,or high tri-expression of α-SMA,Cx43 and LDH-B was positively associated with the TNM stage(p<0.05).Interestingly,high tri-expression of α-SMA,Cx43 and LDH-B was also strongly associated with NSCLC metastasis(p<0.05).7.Survival analysis showed that individual overexpression of α-SMA,Cx43,or LDH-B significantly decreased overall survival(OS)and relapse-free survival(RFS)in NSCLC patients(p<0.05).Moreover,high bi-expression of α-SMA and Cx43,Cx43 and LDH-B,or α-SMA and LDH-B was also associated with lower OS and RFS(p<0.05).Additionally,patients with overexpression of all three molecules had the lowest OS and RFS(p<0.05).univariate and multivariate Cox proportional hazards analyses revealed that only high tri-expression of α-SMA,Cx43 and LDH-B was an independent prognostic factor for OS and RFS in patients with NSCLC(p<0.05).Taken together,these findings demonstrate that the combination of α-SMA,Cx43 and LDH-B levels predicts a poor outcome in NSCLC patients.Conclusions:1.CAFs cells show increased aerobic glycolysis upon the activation of NSCLC cells.2.NSCLC cells shift metabolism to OXPHOS in response to CAFs with aerobic glycolysis through Cx43-formed GJIC.3.High expression of α-SMA,Cx43 or LDH-B are correlated with TNM stage and NSCLC metastasis.High bi-expression of α-SMA and Cx43,Cx43 and LDH-B,or high tri-expression of α-SMA,Cx43 and LDH-B is positively associated with the TNM stage.High tri-expression of α-SMA,Cx43 and LDHB is also strongly associated with NSCLC metastasis.Tri-high expressions of α-SMA,Cx43,and LDH-B predict poor prognosis in NSCLC patients.