The Roles And The Mechanisms Of Angiopoietin-Like Protein 2 In NSCLC Progression Via Regulating Functional Phenotype Of TAMs

BackgroundLung cancer is the leading cause of death from cancer worldwide.Based on histology,Most of lung cancers are non-small cell lung cancer(NSCLC).Although some of the advanced patients benefited from molecular targeted therapies in recent years,drug resistance will always happen.Prognosis of patients is still not optimistic.Studies reported that interactions between tumor cells and TME(tumor microenvironment)play important roles in tumor progression,which makes TME to be a hot area in tumor therapy researches.Exploration of molecular mechanisms underlying NSCLC metastasis and progression from the view of TME can improve the effects of therapies.Angptl2(angiopoietin-like protein 2)is a secretory glycoprotein and is structurally similar to angiopoietins.Physical Angptl2 signaling maintains tissue homeostasis via adaptive inflammation,whereas over activation of Angptl2 signaling causes pathological tissue remodeling.Angptl2 is highly expressed in various cancer types,including NSCLC,and facilitates cancer metastasis by promoting tumor cell invasion and angiogenesis.Previous studies have focused on how tumor cell-derived Angptl2 directly affected tumor progression.Whether the action of Angptl2 on stromal cells is involved in Angptl2-driven tumor behaviors remains unknown.TME is composed of tumor cells,immune cells,vascular and lymphatic endothelial cells and extracellular matrix.These cells interact with each other through growth factors,angiogenic factors and chemokines,further promote tumor proliferation,invasion and distant metastasis.They cooperate to create the most favorable conditions for tumor progression.Bone marrow-derived cells are predominant stromal cells in the TME,which account for about the 15?20%of the total solid tumor.Macrophages that infiltrate tumor tissues are referred to as tumor-associated macrophages(TAMs).They have two well-established phenotypes,classically(M1)-and alternatively(M2)-activated macrophages due to different microenvironment situations.M1 phenotype is characterized by IL-12high,IL-23high,IL-10low,and they produce nitric oxide,inflammatory factors and so on;M2 phenotype is characterized by IL-10high,IL-12low,IL-23low,and they activates tissue repair procedures,angiogenesis and inhibits adaptive immune response.M1 macrophages are viewed as anti-neoplastic while M2 are immunosuppressive and tumor promotive.The two subtypes can convert to each other.How to reduce the M2 polarization of TAMs has become a "charm target" for tumor therapy.This study composed of three parts:1)effects of Angptl2 on TAMs polarization and its molecular mechanism;2)effects of Angptl2-induced TAMs on the biological behaviors of NSCLC cells;3)roles of TAMs play in Angptl2 influenced NSCLC xenografts growth.This research for the first explores the mechanisms of Angptl2 promoting NSCLC from the view of TME,which provides a theoretical basis for the treatment of NSCLC by regulating TAMs phenotype.Research purposes1.To clarify the expression of Angptl2 in primary NSCLC tumor tissue;the relationship between Angptl2 expression and pathological parameters and overall survival of NSCLC patients;2.To clarify effects of Angptl2 on TAMs functional phenotype;3.To clarify the molecular pathways function in Angptl2 induced macrophages;4.To clarify effects of Angptl2-induced macrophages on the proliferation,invasion and migration of NSCLC cells;5.To determine whether Angptl2 influences NSCLC growth in nude mice via TAMs.MethodsFirst part1.The expression of Angptl2 in NSCLC and adjacent lung tissues was detected by IHC;the correlation between the expression of Angptl2 in NSCLC and the clinical pathologic parameters was analyzed by chi-square test.Kaplan-meier was used to analyze the relationship between Angptl2 expression and the overall survival of NSCLC patients.2.The expression of CD68,the molecular marker for macrophages,and CD34,the molecular marker for vascular endothelial cells was detected by IHC,and the correlation between Angptl2 expression and TAMs infiltration and MVD was analyzed by Pearson Correlation Coefficient.3.The expression of Angptl2 in 5 NSCLC cell lines and BEAS-2B was detected by Western blot.H1299 with low Angptl2 expression was transfected by Angptl2 plasmid;A549 with high Angptl2 expression was transfected by Angptl2 shRNA plasmid.4.The THP-1 cells were stimulated by PMA to differentiate into macrophages.5.H1299-MOCK/H1299-Angptl2,A549-shNC/A549-shAngptl2 and THP-1-derived macrophages were co-cultured respectively.The proportion of M2 macrophage(CD206+,CD209+)was detected by flow cytometry.6.RT-PCR was used to detect mRNA expression of molecular markers for M1 macrophages(TNF-?,IL-12)and M2 macrophages(IL-10,Argl)in rAngptl2 induced macrophages.7.Phosphorylation of signal pathway proteins was detected by western blot in macrophages stimulated by rAngptl2;pathway inhibitors were further used to screen downstream pathways.Second part1.Western blot was used to detect protein expression of tumor-promoting cytokines,VEGF-A,MMP-9 and TGF-? in macrophages stimulated by rAngptl2.2.Three groups of CM were collected:rAngptl2 stimulated macrophages;rAngptl2 added into complete culture medium;control solution PBS stimulated macrophages.3.Effects of 3 groups of CM on NSCLC cells proliferation were detected by CCK8.4.Effects of 3 groups of CM on NSCLC cells migration were detected by scratch healing experiment.5.Effects of 3 groups of CM on NSCLC cells invasion were detected by transwell invasion experiment.6.Effects of 3 groups of CM on the tube-formation ability of HUVEC cells were detected by tube formation experiment.7.Effects of 3 groups of CM on HUVEC cells migration were detected by transwell migration experiment.Third part1.H1299-Angptl2 and H1299-MOCK cell lines were established through lenti virus infection.2.Construction of subcutaneous NSCLC transplanted tumor model in nude mouse.3.Nude mouse were divided into 4 groups:H1299-MOCK,H1299-Angptl2,H1299-Angptl2+CCL:tail intravenous injection of CCL,H1299-angptl2+ CNL:tail intravenous injection of CNL.4.Keep a record of the longest and shortest diameters of the transplanted tumors every week;sacrifice the mouse at the fifth week.5.Observe the effects of Angptl2 on the growth of NSCLC transplanted tumor.6.IHC was used to detect the TAMs density(F4/80+)to clarify if CCL led to macrophages apoptosis.7.Observe the effects of Angptl2 on the growth of transplanted tumors in the case of macrophage apoptosis.Research resultsFirst part1.Angptl2 is highly expressed in human NSCLC samples and the expression was correlated with poor overall survivalThe positive expression of Angptl2 protein showed brownish yellow to brown particles,mainly expressed in the cytoplasm of NSCLC cells.There were 58 cases(58/81,71.6%)showing Angptl2-positive expression in NSCLC tissues,but only 16 cases(16/54,29.6%)showing Angptl2-positive expression in adjacent normal tissues.Expression of Angptl2 was positively correlated with tumor size(p=0.022)and higher pathological stage(p=0.037).Moreover,Kaplan-Meier survival analysis showed that the levels of Angptl2 negatively correlated with the overall survival(OS)of NSCLC patients.2.Angptl2 expression was positively correlated with TAMs density amd MVDA Pearson correlation analysis showed that Angptl2 expression was positively correlated with TAMs density(r=0.5848)and MVD(r=0.2399)3.Expression of Angptl2 in NSCLC cellsAngptl2 was detected in all five NSCLC cell lines.H1650 and H1299 cells were low expressed;A549,H226 and H1975 were highly expressed,and BEAS-2B cells had the lowest Angptl2 expression.4.Angptl2 promoted TAMs M2 polarizationTranswell co-culture of H1299-Angptl2 with THP-1-derived macrophages led to an increased percentage of CD206+ and CD209+ macrophages compared with H1299-MOCK macrophages,as detected by flow cytometry.Macrophages co-cultured with A549-shAngptl2 cells had lower percentage of M2 TAMs.5.Induction of M2 polarization of TAMs by AngptI2 was dependent on p65 NF-kB signalingRAngptl2 stimulation increased phosphorylation of Stat6 and p65.Selective inhibitors were used to explore which pathways were involved in Angptl2 promoted M2 macrophage polarization:AS1517499(Stat6 inhibitor)and PDTC(NF-?B inhibitor).PDTC treatment attenuated Angptl2-induced macrophage M2 polarization.Second part1.Angptl2 induces the expression tumor promoted cytokines in macrophages.Western blot showed that Angptl2 induced VEGF-A,MMP-9 and TGF-?expression in macrophages.2.Effects of Angptl2-induced macrophages on biological behaviors of NSCLC cellsCCK8 proliferation test,transwell invasion assay and wound-healing assay showed that rAngptl2-promoted macrophages enhanced proliferation,invasion and migration of NSCLC cells.The promotion was higher than the Angptl2 protein itself.3.Effects of Angptl2-induced macrophage on biological behaviors of HUVEC cellsTube formation assay and transwell migration assay showed that rAngptl2-promoted macrophages enhanced tube formation and migration abilities of HUVEC cells.The promotion was higher than the Angptl2 protein itself.Third part1.Angptl2 promoted NSCLC tumor growth in vivoTumor volume and weight in the Angptl2 up-regulated group were significantly higher than the control group.IHC results also showed that TAMs infiltration and MVD was higher in Angptl2 up-regulated group.2.Angptl2 promoted NSCLC tumor growth in the presence of TAMs in vivoIntravenous tail injections of CCL were used to deplete macrophages.IHC showed that F4/80+ cells density was significantly lower in tumor tisses with CCL injected.Depletion of macrophages impaired the enhancement of tumor weight,volume and MVD induced by Angptl2.Conclusions1.Angptl2 is highly expressed in human primary NSCLC tissues;the expression is associated with poor prognosis of NSCLC patients.2.Angptl2 induces TAMs M2 polarization.3.Angptl2 induces TAMs M2 polarization through the P65 NF-?B signaling pathway.4.Angptl2 promotes several tumor-promoting factors production in macrophages.5.Angptl2-induced macrophages promote the proliferation,invasion and migration of NSCLC cells,as well as the migration and tube formation abilities of HUVEC cells.6.Angptl2 promotes the growth of NSCLC xenografts in nude mice through TAMs.