The Study Of Enhanced Immunogenicity And NK Cell Activity By Gemcitabine In Lung Cancer

BackgroundLung cancer is one of the common malignant tumors.Gemcitabine inhibits DNA synthesis.Its antitumor activity has been proved successfully in clinic.Gemcitabine combined with cisplatin is the first-line chemotherapy for NSCLC,which is superior to cisplatin alone in efficacy,disease progression and overall survival rate.Although gemcitabine can effectively kill cancer cells,many patients still suffer from cancer recurrence after conventional chemotherapy.Cytotoxic chemotherapy drugs also have a variety of immune mechanisms.Therefore,the most effective strategy to prevent cancer recurrence may be to mobilize and stimulate the immune system against tumor cells.The definition of tumor immunogenicity is to induce tumor to produce immune response through various ways to prevent tumor growth.In some anticancer treatments,the dead cancer cells will release immune regulatory factors or damage related molecular patterns(DAMPs).These as"danger signals"enhance the immunogenicity of cancer cells.High mobility group 1 protein(HMGB1)as an endogenous risk signal,dendritic cells(DCs)were recruited to bind to toll like receptor 4(TLR4)to activate T cell immune response.Calreticulin(CRT)can be induced to migrate from the endoplasmic reticulum cavity to the cell membrane by chemotherapy and make it easy to be phagocytized by DCs.Natural killer cell is an important anti-virus and anti-cancer innate immune lymphocyte.As an important activated receptor of NK cells,NKG2D ligands(NKG2DLs)are very important to recognize and clear tumor cells.NKG2DLs are expressed mostly on tumor cells but show low expression on the surface of healthy cells.Increasing expression of NKG2DLs on tumor cells and thus increasing the killing activity of NK cells could enhance anti-tumor immunity effectively.ObjectiveThe purpose of this study is to explore the mechanism and effect of antitumor by detecting the immunogenicity of tumor cells induced by different doses of gemcitabine,so as to provide theoretical basis for the treatment of lung cancer patients with immunocompetent function based on gemcitabine.MethodsImmunofluorescence was used to detect the exposure of immunogenic molecules CRT and HMGB1 after gemcitabine stimulation in LLC cells and A549 cells.Dead staining was used to detect the survival of LLC cells and A549 cells stimulated by chemotherapy.The mRNA and protein expression of NKG2D ligands in LLC cells and A549 cells stimulated by gemcitabine were confirmed by quantitative real-time polymerase chain reaction(qRT-PCR)and flow cytometry respectively.In vivo experiments,LLC subcutaneous tumor bearing mice model was established.Tumor volume and weight were monitored.Gemcitabine combined with cisplatin was used to treat tumor.Blood routine was analyzed by blood routine analyzer after harvest,and the exposure of immunogenic molecules CRT and HMGB1 of subcutaneous tumor was detected by immunofluorescence.Flow cytometry was used to detect immune cells and functional molecules in mouse spleens.The spleen NK cells of C57BL/6mice kill the untreated LLC cells and gemcitabine preliminary stimulated LLC cells by carboxyfluorescein diacetate N-succinimidyl ester(CFSE).NK cells purified from mouse spleens after chemotherapy treatment kill the untreated LLC cells by real-time cellular analysis(RTCA).H&E staining of liver and kidney was used to compare the toxic effects of different treatments.ResultsImmunofluorescence results suggested that CRT membrane expression and HMGB1 exposure were increased after gemcitabine treatment in LLC cells and A549cells.Low-dose gemcitabine treatment led to more CRT and HMGB1 fluorescent cells exposure than high-dose gemcitabine.High-dose gemcitabine causes more LLC cells and A549 cells death in Dead staining experiment.The qRT-PCR showed that gemcitabine stimulation induced upregulation of expression of NKG2D ligands mRNA levels in LLC cells and A549 cells.Gemcitabine induces NKG2D ligands expression in protein levels of gemcitabine-treated A549 cells by flow cytometry.In vivo,immunofluorescence showed that mean fluorescence intensity(MFI)per square centimeter of CRT and HMGB1 in subcutaneous tumor treated with low(30 mg/kg)-dose gemcitabine was higher than that of the medium(60 mg/kg)-and high(120mg/kg)-dose gemcitabine groups.Compared with the control group,there was no significant change in the absolute number of lymphocytes of blood count in low-,medium-and high-dose gemcitabine-treated mice,but the lymphocyte percentage was increased.In all chemotherapy treatment groups,the absolute number and percentage of neutrophils of blood count declined.In mice spleen flow cytometry assays,the percentage of NK cells increased in the low-dose group,but not in the medium-and high-dose groups.The expression of NKG2D in mice of NK cells was up-regulated in all the gemcitabine treated groups.The expression of IFN-γin NK cells increased after low-dose gemcitabine treatment.The MFI and percentage of Ki67 in the high-dose gemcitabine group were lower than those in the PBS group.The results of CFSE showed that the lung cancer cells stimulated by low-dose gemcitabine were more sensitive to NK cell killing.RTCA results showed that the cytotoxicity of NK cells in all gemcitabine treated group was higher than that in the control group.The cytotoxicity of NK cells in the low-dose gemcitabine group was stronger than that in the medium-and high-dose groups.The inhibitory effect of all gemcitabine-treated groups on the tumor volume was significantly higher than that of PBS group.All gemcitabine-treated groups had no effects on body weight.H&E staining of the liver showed vacuolar degeneration with increasing gemcitabine dose,but excessive kidney damage was not observed.ConclusionsIn this study,we observed that low-dose gemcitabine treatment increased the membrane expression of CRT and the exposure of HMGB1,upregulated expression of NKG2D ligands,activated NK cells and enhanced antitumor immunity.Low-dose gemcitabine treatment has no obvious toxicity to the immune system and triggers anti-tumor effect by enhancing immunogenicity.We got the following conclusions:1.Low-dose gemcitabine enhances exposure of immunogenic molecules in lung cancerLow-dose gemcitabine treatment can enhance the exposure of lung cancer cells to immunogenic molecules CRT and HMGB1 in vitro cells and in tumor-bearing mice.High-dose gemcitabine causes lung cancer cell death.2.Gemcitabine induces upregulation of innate immune system-related immunogenic molecules in lung cancerGemcitabine can increase the expression of NKG2D ligands in human and mouse lung cancer cells,thereby increasing the anti-tumor immunity of NK cells.3.Low-dose gemcitabine induces activation of NK cells in a mouse model of lung cancerGemcitabine reduced the absolute number of neutrophils in the blood of mice.The expression of IFN-γin spleen NK cells of mice treated with low-dose gemcitabine increased,while Ki67 decreased in high-dose gemcitabine group.Low-dose gemcitabine activates the NK cell immune system,while high-dose gemcitabine inhibits NK cell proliferation.The level of IFN-γproduced by NKG2D~+NK cells is higher than NKG2D~-NK cells.Low-dose gemcitabine enhanced the NK cell immune response in mouse models of lung cancer.4.Low-dose gemcitabine treated lung cancer cells are more sensitive to NK cell killingNK cells have a stronger killing effect on LLC cells stimulated by low-dose gemcitabine.NK cells of spleen were more sensitive to lung cancer cells after low-dose gemcitabine treatment.Gemcitabine had no direct effect on the expression of Ki67,NKG2D and IFN-γin mouse spleen NK cells.5.Low-dose gemcitabine treatment inhibits the growth of lung cancer in miceLow-dose gemcitabine inhibites tumor growth in mice and has no significant adverse effects on the liver and kidney tissues of mice.