The Effect Of Tumor-associated Macrophages On Development And Drug Resistance Of Lung Adenocarcinoma

PART I INDUCTION AND PHENOTYPIC IDENTIFICATION OF TUMOR-ASSOCIATED MACROPHAGESObjective: to establish human tumor-associated macrophages(TAM)in vitro,and analyze their biological phenotype.Methods: It is generally believed that the normal macrophages are M1 type macrophages,and TAM are M2 type macrophages.In this experiment,we first stimulated human monocyte cell line THP-1 cells into macrophages(PMA only)by phorbol-12-myristate-13-acetate(PMA).The flow cytometry was used to analyze the expression level of CD14 on the surface of macrophages(PMA only);The human interleukin 4(IL-4)or lipopolysaccharide(LPS)was used to polarize macrophages into M2 or M1 macrophages;Semi-quantitative RT-PCR was used to analyze the expressions of IL-6 m RNAs in different types of macrophages;the protein levels of TNF-? were detected by the ELISA in different types of macrophages.Results: The expression of CD14 was significantly increased in THP-1 cells induced by PMA.Compared with that in M1 macrophages induced by LPS alone,the PMA-induced macrophages(PMA only)and M2 macrophages induced by IL-4 had the lower expression levels of IL-6 m RNA and TNF-? protein.Conclusions: Macrophages were successfully induced and showed high expression of CD14.The PMA-induced macrophages(PMA only)had the tendency to differentiate into M2 macrophages.TAM was successfully induced and had the phenotype of M2 macrophages,which was characterized by low expressions of IL-6 and TNF-?.PART II THE EFFECT AND PRELIMINARY MECHANISM OF TUMOR-ASSOCIATED MACROPHAGES ON MIGRATION AND INVASION OF LUNG ADENOCARCINOMA CELLObjective: To investigate the effect and the underlying molecular mechanism of tumor-associated macrophages(TAM)on migration and invasion of human lung adenocarcinoma A549 cells.Methods: The A549 cells and tumor-associated macrophages(TAM)were cocultured in Transwell chamber.The cocultured fluid was collected for subsequent experiments.The wound-healing assay and Transwell assay were used to detect the migration and invasion of A549 cells in tumor-associated macrophage microenvironment;Semi-quantitative RT-PCR and q RT-PCR and western blot were used to detect the effect of tumorassociated macrophages(TAM)on the expressions of VEGF-C ?VEGF-D and VEGFR3 on A549 cells;IHC was used to detect the distribution of VEGFR3 in vivo.In the tumor-associated macrophage(TAM)microenvironment,The wound-healing assay and transwell assay were used to detect the migration and invasion ability of A549 cells after the treatment of VEGFR3 inhibitor(MAZ51),respectively.Results: Tumor-associated macrophages(TAM)promoted the migration and invasion of lung adenocarcinoma A549 cells.The VEGFR3 was mainly expressed on tumor cells.Tumor-associated macrophages(TAM)promoted the expressions of VEGF-C,VEGF-D and VEGFR3 in A549 cells.Inhibition of VEGFR3 reduced the migration and invasion of lung adenocarcinoma A549 cells in the tumor-associated macrophage(TAM)microenvironment.Conclusions: Tumor-associated macrophages(TAM)promoted the migration and invasion of A549 cells by increasing the expression of VEGFR3 in A549 cells.PART ? EFFECT OF TUMOR-ASSOCIATED MACROPHAGES MEDIATED VEGFR3 ON TUMOR PROLIFERATIONObjective: To investigate the effect and the underlying molecular mechanism of tumor-associated macrophages mediated VEGFR3 on the proliferation of lung adenocarcinoma cells.Methods: First,the cell viability of cocultured A549 cells was detected by MTT in different concentrations of VEGFR3 inhibitor(MAZ51);flow cytometry was used to detected the apoptosis of cocultured A549 cells in the presence of MAZ51;cell-colony formation was used to analyse the inhibitory effect of MAZ51 on the proliferation of cocultured A549 cells;western blot assay was used to analyse the MAPK signaling pathway and P53,PTEN,BCL-2 and MMP-2 expressions of cocultured A549 cells in the presence of MAZ51;semi-quantitative RT-PCR was used to detect the expressions of P53 and PTEN m RNA of cocultured A549 cells in the presence of U0126(ERK signaling pathway inhibitor)and si RNAs against P53 and PTEN.MTT assay was used to analyze the activity of MAZ51-treated cocultured A549 cells after the treatment with si RNAs against P53 and PTEN.Results: MAZ51 inhibited the activity of cocultured A549 cells,promoted the apoptosis of cocultured A549 cells and decreased the number of cell-colony formations.Western blot showed that MAZ51 could inhibit the activity of p-ERK,increase the expressions of tumor suppressor proteins P53 and PTEN and decrease the expressions of BCL-2 and MMP-2.After inhibition of ERK signaling pathway,the expressions of P53 and PTEN m RNA were significantly increased,After the treatment of si RNAs against P53 and PTEN,the proliferative activity in MAZ51-treated cocultured A549 cells was significantly improved.Conclusions: VEGFR3 inhibition could decrease the activity of p-ERK to improve the expressions of tumor suppressor proteins P53 and PTEN,thus decreasing the activity of lung adenocarcinoma cells.PART ? EFFECT OF TUMOR-ASSOCIATED MACROPHAGES MEDIATED VEGFR3 ON CHEMOSENSITIVITY OF TUMOR CELLSObjective: To investigate the effect of tumor-associated macrophages mediated VEGFR3 on chemosensitivity of lung adenocarcinoma cells,thus providing a new therapeutic strategy for the treatment of lung adenocarcinoma.Methods: Flow cytometry was used to detect the apoptosis ratio of cocultured A549 cells after the treatments of various combinations of VEGFR3 inhibitor(MAZ51)and doxorubicin in vitro.In vivo,a subcutaneous tumor model of nude mice was constructed to detect the effect of VEGFR3 inhibitor on chemosensitivity of lung adenocarcinoma cells.Results: Compared with that after the use of MAZ51 alone,doxorubicin alone or two drugs simultaneously,Pretreatment with Maz51 followed by doxorubicin had the largest proportion of apoptosis in vitro.In vivo,the impact of Maz51 in combination with doxorubicin chemotherapy on cocultured A549 cells resulted in a significant reduction in tumor growth compared to doxorubicin therapy alone.Conclusions: Maz51 could enhanced the chemosensitivity of doxorubicin in lung adenocarcinoma.