Characterizing Membrane Lipid Phenotypes Of Macrophage In Different Lung Cancer Microenvironments Using Mass Spectrometry

Background and Purpose:In the cancer microenment,macrophages are affected by lung cancer cells and their cytokines then make corresponding immune responses.The function of macrophages is complex and have alternative effect to anticancer or promoting cancer function.In anticancer terms,macrophages play the role of Ml macrophages in phagocytosis,the release of anticancer cytokines,the role of antigen presenting and the initiation of adaptive immune respuonse.Promoting cancer function is mainly manifested in inhibiting the immune response of T cells,repairing damaged tissues incorrectly,promoting angiogenesis in solid tumor and protecting tumor stem cells.Macrophages start function on the basis of metabolic changes,in which metabolites play the role of reactive substrates and signaling pathway activator.This study use mass spectrometry arnalysis for metabolomics to obtain obvious change characteristics of metabolites from macrophages in lung cancer,and use related biological technology to obtain the macrophage activation parameters to evidence it.Methods:The monocyte/macrophage RAW264.7 was stimulated by various lung tumor cell lines(adenomatous cell line A549,squamous cell line H157,and undifferentiated large cell line H460)-derived culture supernatants,establishing an imitative tumor microenvironment as well as normal lung tissue cell line BEAS-2B-derived culture supernatant represented a normal control and the DMEM medium as a blank control.M0 macrophages were cultured for 24h,48h and 72h using a mixed medium containing 20%,50%and 80%culture supernatants,then macrophages were labeled with monocyte/macrophage marker CD14,M1 macrophage marker CD16/32 and M2 macrophage marker CD206 for detection by flow cytometer.In order to further investigation,M0 macrophages with lipopolysaccharide and interferon gamma(the M1 macrophages inducers)in containing 80%culture supernatants for 48 h incubation were set as parallel experimental groups.Then marker CD16/32 was detected by flow eytometer,The cell vitality was CCK-8 cell viability assay kit;Tumor necrosis factor alpha concentration was detected by enzyme linked immunosorbent assay kit;And in situ detection of membrane lipid from macrophages on cell slides(macrophages in the same treatment were cultured in three dishes with slides and three mass spectrum were collected from each slide)was performed in negative ion mode with high resolution(530,000 at m/z 400)mass spectrometry 9.4 T apex-ultraTM hybrid Qh-FTICR MS with matrix-assisted laser desorption/ionization.Then mass spectrometry data was imported to the metabonomics analysis website(MetaboAnalyst 3.0,http://www.metaboanalyst.ca/)and the partial least squares discriminant analysis and hierarchical cluster analysis were made.Identification of nearly 300 metabolites were matched through searching databases and through secondary spectrum analysis.Research Results:1)The qualitative results of flow cytometry showed that different lung cell-derived culture supernatants reduced the level of CD 16/32 expressicon.Moreover,the degree of inhibition was increased with the increase of concentration and stimulation time.The degrees of inhibition of different treated Ml macrophage surface membrane protein CD 16/3 2 was different;2)The relative quantitative results of the CCK-8 cell viability assay of different treated macrophages showed that the different lung cell-derived culture supernatants enhoanced the viability of these macrophages(p<0.01)The cell viability of M1 macrophages were stronger than that of M0 macrophages in DMEM medium(p<0.01).After some stimulation of same culture supernatants,the cell viability of M0 and M1 macrophages was no significant difference(p>0.05);3)The absolute quantitative results of enzyme linked immunosorbent assay showed that the diffrent lung cell-derivd culhture supernatants promoted the large amount of tumor necrosis factor alpha released to anti-cancer function(p<0.01).But there was no significant effect on Ml macrophages(p>0.05).After the stimulation of same culture supernatants,tumor necrosis factor alpha from M0 macropahges and that from M1 macrophages were significant difference(p<0.01).4)Approximately 300 of membrane lipids of each type of macrophages in the five different mediums were detected in negative ion mode through the high-resolution mass spectrometry(530,000 at m/z 400).The partial least square-discriminant analysis of the detected data indicated that PE-Cer(d36:1)contributed the most explanation of component 1,a significant feature to distinguish the treated macrophages associated with the microenvironment.The result of hierarchical clustering analy sis on the basic of top 30 VIP(variable importance for the nprojectionl metabolites suggested that the differences of significant membrane lipid phenotype among the treated macrophages were mainly caused by the different inducers,which were classically activated macrophage directed inducers and the different lung cell-derive culture supernatants.Hierarchical clustering line showed that after the stimulation of normal lung tissue cell-derived culture supernatant,MO macrophages were similar with M0 acrophages cultured in DMEM medium in terms of metabolites,whereas Ml macrophages were similar with M1 macrophages stimulated with lung cancer cell-derived culture supernatants in terms of metabolites.Conclusions:Various hung cancer microenvironments have different effects on macrophages.The difference of membrane lipids between M0 macrophages and M1 macrophages was greater than that of culture supernatant stimulation,and the marked change marker PE-Cer(d36:1)was found.Different lung cancer microenvironment could inhibit the expression of membrane protein CD16/32,but it could enhance the cell viability of macrophages.Different lung cancer microenvironment promoted the release of tumor necrosis factor by MO macrophages,but the effect on Ml macrophages was not significant.