Tumor-Associated Macrophages Induce Gefitinib Resistance In Lung Adenocarcinoma Cells And Its Mechanism

Background and objective:Tumor microenvironment(TME)is the internal environment for the survival and development of cancer tissue,which has become one of the hot spots in cancer research in recent years.Macrophages in tumors are important components of TME and are called tumor associated macrophages(TAMs).Clinical studies have confirmed that TAMs infiltration is associated with the efficacy of epidermal growth factor receptor-tyrosine kinase inhibitors(EGFR-TKIs)in non-small cell lung cancer(NSCLC),but the specific mechanism remains unclear.Therefore,we intend to explore the role of TAMs in NSCLC molecular targeting therapy and its possible mechanism at the cellular level.Methods:1.The polarization of THP-1 cells to macrophages was stimulated by Phorbol-12-myristate-13-acetate(PMA),IL-4 and IL-13.The expression of IL-10 induced to form TAMs was detected by ELISA,and the expression of CD14 and CD163 on the surface of THP-1 was detected by flow cytometry to prove the successful establishment of TAMs model in vitro.2.CCK-8 method was used to detect the effective killing concentration and time of gefitinib on lung adenocarcinoma cell lines A549 and PC-9.The indirect contact co-culture mode of TAMs with lung adenocarcinoma cell lines in vitro was established by Transwell chamber culture method.CCK-8 and EDU proliferation assay were used to detect the effect of TAMs on the proliferation activity of cancer cells.Western Blot was used to detect the effect of TAMs on the expression of key proteins in PI3K/AKT signaling pathway of cancer cells.Results:1.After induction,the growth characteristics of THP-1 cells changed from suspension to adherence,and their morphology changed from round to shuttle,and no longer had proliferative activity.Flow cytometry results also showed that theproportion of double positive expression of CD14 and CD163 in the induced cells was significantly increased.ELISA results showed that the expression level of TAMs IL-10 was significantly increased.Thus,a model of TAMs in vitro was successfully established in this study.2.CCK-8 results showed that the survival rates of lung adenocarcinoma A549 and PC9 in Jilin drug group were 47.67%±3.79%,50.33%±2.52%,95.33%±1.53%,94.67%±3.06% in TAMs group and 72.33%±3.06%,79.00%±3.00% in combination group,respectively.Compared with drug group,the survival rates of lung adenocarcinoma A549 and PC9 cells in combination group were significantly higher.Rise.The results showed that TAMs could increase the survival rate of lung adenocarcinoma cells treated with gefitinib.The results of EDU proliferation test showed that the proliferation rates of lung adenocarcinoma A549 and PC9 in normal culture were 24.97%±1.55% and 21.53%±1.10%,respectively.The proliferation activities of tumor cells in drug group were 6.53%±0.80% and 5.47%±0.60%respectively.The proliferation activities of tumor cells in TAMs group were22.97%±0.70% and 20.03%±0.71%,respectively.The proliferation activities of tumor cells in combination group were 16.63%±0.86% and 14.43%±0.93%,respectively.In TAMs group,there was no significant change in the proliferation activity of tumor cells compared with the control group.The proliferation activity of tumor cells in drug group was significantly decreased,indicating that gefitinib had significant killing effect on lung adenocarcinoma A549 and PC9 cells.The proliferative activity of tumor cells in combination group was significantly higher than that in drug group,and the difference was statistically significant.It is shown that TAMs can reduce the killing effect of gefitinib on tumor cells.3.The results of phospho-PI3 K and phospho-AKT signal detection showed that both control group and TAMs group had higher phosphorylation levels in lung adenocarcinoma A549 and PC9 cells.After treatment with gefitinib,the phosphorylation level in drug group decreased significantly(P<0.05),which confirmed that gefitinib exerted its killing effect on tumor cells by blocking PI3K/AKT signal pathway.In combination group,the expression levels of phospho-PI3 K and phospho-AKT were higher than those in drug group(P<0.05),suggesting that TAMs may affect the tolerance of tumor cells to gefitinib by regulating the phosphorylation levels of PI3 K and AKT in tumor cells.Conclusions:1.Successfully stimulate THP-1 cells to form TAMs by PMA,IL-4 and IL-13,and establish an in vitro study model of TAMs;2.TAMs can reduce the cytotoxicity of gefitinib on lung adenocarcinoma A549、PC9 cells,and its mechanism may be related to the activation of PI3K/AKT signaling pathway.