The Impacts And The Underlying Mechanisms Of Apatinib On The Amino Acid Metabolism And The Immune Microenviroment Of Non-small Cell Lung Cancer

Part one: The effect and mechanism of apatinib on amino acid metabolism of non-small cell lung cancer cellsObjective: Exploring the effects and underlying regulatory mechanism of apatinib on amino acid metabolism of non-small cell lung cancer.Methods: The effect of apatinib on the proliferation of human non-small cell lung cancer cell lines A549 and H460 were evaluated by CCK8,Ed U test and colony forming assay.Cellular apoptosis and cell cycle distribution were tested by flow cytometry.Cyto-ID kit,western blot,GFP-LC3 plasmid transfection and transmission electron microscopy were used to test cellular autophagy level.GC-FID and LC-MS were performed to detect the intracellular amino acid level.2-NBDG uptake test were used to exam the glucose uptake ability.Seahorse test were applied to evaluate glycolysis rate.RNA-seq were used to filtrate relative regulatory pathways.The key regulatory gene ATF4 was knocked down with si RNA to confirm its regulation of downstream genes.The ATF4-silence-H460 cells and negative-control-H460 cells were used to establish xenograft model on nude mice.We tested the synergetic effect of apatinib and ATF4-silence based on the growth curve of xenograft.Results:(1)Cell growth of NSCLC cells were significantly decreased after application of apatinib.The anti-proliferation effect was time-and concentration-dependent.Apatinib of medium and high concentration could induce apoptosis in the tumor cell while the low dose could not.Apatinib could significantly induce cellular autophagy in a concentration depend manner but have no effect on the cellular cycle distribution.(2)RNA-Seq analysis showed that apatinib significantly affected genes and pathways related to metabolism of A549 cells.(3)Apatinib has no significant effect on the glucose uptake and glycolysis level.(4)Apatinib significantly suppressed the expression level of glutaminase 1 (GLS1,an enzyme that catalyzes glutamine to hydrolysis into glutamate)and enhanced the expression levels of asparagine synthetase(ASNS,an enzyme that catalyzes the reaction of glutamine with aspartic acid to generate glutamate and asparagine)and SLC1A5(a glutamine transporter),while it didn’t affect m TOR activity.(5)The intracellular concentrations of glutamine and asparagine were increased after apatinib treatment,and the glutamine concentration decrease at first and increase at later time.Meanwhile,the aspartate concentration was decreased.(6)Apatinib significantly activated the amino acid response pathway.When ATF4 were knocked down,apatinib couldn’t improve the expression levels of ASNS and SLC1A5,and m TOR activity was inhibited(7)The autophagy and apoptosis levels were increased with apatinib treatment when ATF4 was knocked down.The intracellular concentration of glutamine was increased and concentration of glutamate was decreased,while the concentrations of asparagine and aspartate were not changed.(8)In vivo study showed that low concentration of apatinib has no inhibitory effect on the subcutaneous xenograft established with H460 cell.And apatinib has synergetic inhibition effect on tumor growth with ATF4 silence.Conclusion:(1)Apatinib could induce autophagy and inhibit proliferation by decreasing intracellular concentration of aspartate in NSCLC cells.(2)Apatinib decreased intracellular concentration of aspartate that activated AAR pathway and increased the expression levels of SLC1A5 and ASNS,which promoted the uptake and metabolism of glutamine.These might be the reason of poor efficacy or drug resistance of apatinib.(3)ATF4 might maintain the activation of m TOR signal by regulate glutamine metabolism in NSCLC cells.(4)ATF4 knockdown enhanced apatinib’s growth inhibition effect on NSCLC.Part two: The effect and mechanism of apatinib on the immune microenvironment of lung cancerObjective: To exploring the effect of apatinib on the immune microenvironment of lung cancer and the related regulatory mechanism.Methods: In this study,the inmmunodeficient nude mice and C57BL/6 mice were used to establish human and mice subcutaneous xenograft model.Mice were given saline or apatinib with intragastric administration.Flow cytometry was used to test the ratio and function of CD8~+ T cells in the tumor and peripheral blood.The ratios of macrophage,MDSC,DC and NK cells were also investigated.Neutralizing antibodies were used to clear the CD3~+,CD8~+ T cells and clodronate liposomes were used to clear the macrophages of mice.The mice then were given saline and apatinib intragastrically and the growth rates of transplanted tumor were observed.Macrophage cell lines ANA,RAW264.7 and BMDM were polarized to M1/M2,and cells were treated with apatinib.Flow cytometry was used to evaluate the expression levels of M1/M2 phenotypes.RT-PCR was used to detect expression levels of cytokines related to both polarizations.LDH test kit was used to asses cellular apoptosis.NADPH/NADP and GSH/GSSG test kit to exam the ability of oxidation deoxidizing.Western blot and transmission electron micrograph were used to evaluate autophagy level.Immunomagnetic heads were used to isolate CD8~+ T cells from mouse spleen.The cells were incubated directly with apatinib or co-cultured with apatinib treated M2 type BMDM cells,and flow cytometry was used to investigate the function and viability of CD8~+ T cells.Results:(1)Apatinib showed no inhibition on tumor growth for xenograft established by both human lung cancer cell line H460 and mouse lung cancer cell line Lewis on nude mice.(2)Apatinib could significant inhibit growth of tumors transplanted with Lewis cells on C57BL/6 mice.(3)Apatinib treatment didn’t change the CD8~+ T cell ratios,as well as its PD-1 and CTLA-4 expression levels in the tumor or peripheral blood,in C57BL/6 mice which carried xenograft of Lewis cells.However,the excretion level of IFN-γ of CD8~+ T cells was increased.(4)The ratios of MDSCs,DCs and NK cells and the excretion level of IFN-γ of NK cells in the xenograft of Lewis cells has not changed after apatinib treatment in C57BL/6 mice.(5)Apatinib couldn’t inhibit xenograft tumor growth after the in vivo clearance of CD3~+ T or CD8~+ T cells.(6)In vitro test showed that patatinib could increase PD-L1 level in NSCLC cells.However,in vivo test showed PD-L1 level in tumor of both nude mice and C57BL/6 mice has no significant change after apatinib treatment.(7)After apatinib treatment,the ratio of M2 macrophages significantly decreased.(8)In vitro,apatinib reversed the M2 polarization,including downregulation of CD206,Arg1 and IL-10 expression levels,and upregulation of CD86,Nos2 and IL-12.(9)Apatinib maintained the redox balance of M2-polarization macrophages in vitro,which included the increasing of intracellular ROS,NADPH/NADP ration and GSH/GSSG ration.(10)Apatinib inhibited the proliferation of M2-polarization macrophages in vitro,but didn’t kill cell,which appeared as it didn’t change the secretion of LDH.(11)Apatinib could increase autophagy level in M2 macrophage.When the autophagy was inhibited,the reversal effect of apatinib on M2 phenotypes were also suppressed.(12)Directly treatment with apatinib led to the inhibition of cellular viability and IFN-γsecretion of CD8~+ T cells.However,the viability and IFN-γexcretion of CD8~+ T cells increased after co-cultured with apatinib treated BMDM-M2 cells.(13)The inhibitory effect of apatinib on the growth of transplanted tumors were suppressed after macrophage clearance.Conclusion:(1)The tumor suppression effect of apatinib was CD8~+ T cells and macrophages dependent.(2)Apatinib promoted the activation of CD8~+ T cells through reversing the M2 polarization of macrophages.(3)Apatinib inhibited the proliferation of M2-polarization macrophages and increased the production of ROS,but had no significant cytotoxic effect to macrophage,which might due to its promotion of cellular reductive ability.(4)Apatinib could induce cellular autophagy through inhibiting m TOR signal,which also contributed to revering M2 polarization of macrophages.