Effect Of Immune Microenvironment Modulation On EGFR-TKI Inhibitory Function In Heterogeneous Lung Cancer

Globally,lung cancer is by far the leading cause of death in all types of cancer.There are various new approaches for treating lung cancer despite traditional surgery,chemotherapy and radiotherapy.While for the advanced lung cancer,the survival odd is still less than 5% in 5 years.Therefore the exploration for the new treatment is imperative.Molecular targeted therapy was newly developed cancer therapy in recent decade,since Epidermal Growth Factor Receptor(EGFR)mutation were identified as driving gene of cancer,EGFR pathway targeted therapy now becomes key area in precise treatment of lung cancer.As two prominent examples of EGFR-TKI,Gefitinib and Erlitinib have been widely used for advanced or metastatic NSCLC with poor response to chemotherapy or radiotherapy.However,multiple perspective clinical studies shows a large proportion of NSCLC lung cancer patients would become resistant to Gefitinib after 9-13 months of treatment.So,understanding the underlying mechanisms become the research priority to overcome this drug resistant problem.EGFR-TKI induced apoptosis of EGFR highly-expressed lung cancer cells through upregulating BIM.Apoptosis of cancer cells becomes prevented as lung cancer turn to be resistant to EGFR-TKI,this phenomenon is accompanied with down regulation of two key molecules in apoptosis pathway,BIM and FOXO-3a.Therefore,BIM as target gene in cancer treatment draws increasingly research attention and now has been a new research hot spot,for the upregulation of BIM may relate to apoptosis of cancer cells.Data in recent years has also showed that response to EGFR-TKIs therapy are closely related with BIM and FOXO-3a expression in tumor.For the patients whose tumor cannot be completely eliminated by standard clinical treatment,the application of immunotherapy can be a valuable alternative approach.Immunotherapy is relatively less toxic and can prolong the survival time of patients and improve the life quality of patients.It provides a new direction for the treatment of patients with lung cancer and a promising research field of tumor therapy more than ever.Tumor microenvironment determines the results of anti-tumor response of host's immune systems.Cytokines in tumor microenvironment plays vital role in determine the status of tumor microenvironment,the tumor microenvironment can be tuned by changing the cytokines associated with T helper cells.It is reported that IFN-? secreted by CIK reversed DDP resistance of A549/DDP cell through down regulating GST-7,which indicated that there were potential links between cytokines in tumor immune microenvironment and drug resistance of lung cancer.However,the question that whether and how tumor immune microenvironment modulation can effect tumor drug resistance,or its modulation can contribute to inhibitory effect of EGFR-TKIs remains to be answered.In this study,using in vitro and in vivo methods,lung cancer cell lines with different sensitivity to EGFR-TKIs(EGFR-TKIs sensitive type,wild type,EGFR-TKIs resistant type)are used to investigate the effect of immune microenvironment modulation on EGFR-TKI inhibitory function and underlying mechanism,aiming to provide some theoretical basis for better comprehensive treatment plan for EGFR-TKI resistant lung cancer.Objective:To investigate the effect of DC-CIK and its supernatant on lung cancer lines with different sensitivity to EGFR-TK;and the influence of specific immune microenvironment(specific DC-CIK supernatant)modulation on the treatment of lung cancer with different sensitivity to EGFR-TK together with molecular targeted drug EGFR-TKI gefitinib and the underlying mechanism.Methods: Part1: By applying flow cytometry,we compared the phenotype markers of control DC-CIK and specific DC-CIK,and then compared the supernatant of control DC-CIK and specific DC-CIK in levels of IFN-??TNF-??IL-2.The morphological character and targeted effect of specific DC-CIK to lung cancer cells(EGFR-TKIs sensitive type,wild type,EGFR-TKIs resistant type)were observed with light and electron microscopy.Part2:(1)Effect of gefitinib,specific DC-CIK supernatant and the combination to three lung cancer cells(EGFR-TKIs sensitive type,wild type,EGFR-TKIs resistant type)were observed with light a microscopy.The inhibitory effects of gefitinib,specific DC-CIK supernatant and combinations of two on the expression of three lines of lung cancer cells were compared via the optical microscope observation and MTT experiment.(2)The expression of BIM and FOXO-3a in three lung cancer cells were measured by real-time quantitative PCR and Western Blot.(3)The therapeutic effect of gefitinib,specific DC-CIK supernatant and combinations of two in the treatment of lung cancer using tumor bearing nude mice,with measuring the expression of BIM and FOXO-3a by immunohistochemistry.Part3:(1)The inhibitory effects of EGFR-TKI gefitinib,specific DC-CIK supernatant and combinations of DC-CIK supernatant and gefitinib on(A549 and H292?Hcc827 and Hcc827)mixed cell were measured by MTT.(2)The the inhibitory effects of 2.5?M,5?M,10?M gefitinib on H292 cultured with A549 supernatant?Hcc827supernatant and wild type A549 were measured by MTT.The expression of BIM and FOXO-3a in mixed lung cancer cells were measured by real-time quantitative PCR.(3)The therapeutic effect of gefitinib,specific DC-CIK supernatant and combinations of two using nude mice bearing heterogeneous tumor with mixed cells were measured,and the expression of BIM and FOXO-3a in tumor were measured by immunohistochemistry.Results: Part1: The results of flow cytometry showed that the level of CD3+CD8+,CD3+CD56+ of the specific DC-CIK of was higher than that of the control DC-CIK,the concentration of IFN-,TNF-and IL-2 in the supernatant of the specific DC-CIK was higher than that of the control DC-CIK.Part2: Microscopically,the specific DC-CIK cells can be targeted to tumor cells and the tumor cells can be lysed by CIK.The MTT experiment showed that the specific DC-CIK supernatant combined with gefitinib had stronger inhibitory effect on lung cancer cells than that of gefitinib alone or DC-CIK alone.Expression of BIM and FOXO-3a detected by real-time quantitative PCR and Western Blot in lung cancer cells were higher in the combined treatment group than that in the gefitinib group alone or in the specific DC-CIK supernatant group.The results of nude mice bearing tumor showed that the tumors in the combine treatment groups were the smallest.The results of immunohistochemistry showed that the expression of apoptosis marker Caspase-3 in the two combined treatment group were significantly higher than that in other groups,and proliferation marker Ki-67 were significantly lower in combined treatment group.Part3: The inhibition rate of gefitinib on A549 supernatant ?Hcc827 supernatant cultured H292 and wild type H292 are measured by MTT,the results showed that the A549 supernatantcultured H292 were less sensitive to gefitinib than wild type H292?the Hcc827 supernatantcultured H292 were more sensitive to gefitinib than wild type H292.The inhibition rate specific DC-CIK supernatant on three different combination of heterogeneous lung cancer cells are measured by MTT.The specific DC-CIK supernatant combined with gefitinib had strongest inhibitory effect on heterogeneous lung cancer cells(H292+A549?H292+Hcc827).Expression of BIM and FOXO-3a detected by real-time quantitative PCR in lung cancer cells were higher in the combined treatment group than that in the gefitinib group alone or in the specific DC-CIK supernatant group.The results of nude mice bearing tumor with heterogeneous cancer cells showed that the tumors in the combine treatment groups were the smallest.The results of immunohistochemistry showed that the expression of apoptosis marker Caspase-3 in the two combined treatment group were significantly higher than that in other groups,and proliferation marker Ki-67 were significantly lower in combined treatment group.Conclusions: Lung cancer cells can be targeted and collectively lysed by the specific DC-CIK.Level of IFN-??TNF-??IL-2 in supernatant of specific DC-CIK are higher than that in control DC-CIK.Modulation the tumor immune microenvironment has a synergistic effect on gefitinib in inhibiting lung cancer cells with different sensitivity of gefitinib and heterogeneous lung cancer cells both in vitro and in vivo,and its underlying mechanism is related to the up expression of BIM and FOXO-3a.