The Impact Of Myeloid Derived Suppressor Cells Autophagy On Lewis Lung Carcinoma Tumor-bearing Mice

Objective Observed the impacts of Lewis lung carcinoma(LLC) cell line derived cell culture supernatant(tumor cell-conditioned medium, TCCM) on the autophagy of myeloid myeloid derived suppressor cells(granulocytic myeloid-derived suppressor cells, G-MDSC); Study the effect of autophagy on G-MDSC survival and function under tumor microenvironment; research the effect of G-MDSC autophagy on the development of Lewis transplant tumor in mice.Methods 1. Isolation and identification of G-MDSC Constructed Lewis transplant tumor in mice,G-MDSC were obtained with MACS sorting method from the spleen of tumor-bearing mice, after the fluorescent antibody-labeled cell surface marker molecules CD11 b and Ly-6G/Ly-6C, flow cytometry(FCM) detected its purity; the isolated cell smears stained with Wright-Giemsa stain, cell morphology was observed under optical microscope. 2. Preparation of cell culture supernatant of Lewis lung carcinoma After Lewis cells cultured with DMEM within high glucose medium containing 10% NBCS(newborn calf serum), until the degree of cells polymerization reached 90%, replaced with serum-free DMEM culture medium, cultured for 24 hours, the supernatant was collected. 3. TCCM inhibit the apoptosis and promote the ROS generation of G-MDSC G-MDSCs were treated with different concentrations of TCCM for 12h/24 h,then stained with Annexin V / PI dye or DCFDA,then detected the influence of TCCM on cell apoptosis and the level of cell effector molecule of ROS by FCM. 4. TCCM promote G-MDSC autophagy G-MDSCs were cultured in containing 10% FBS(fatal bovine serunm) RPMI 1640 medium within TCCM and chloroquine(Chloroquine, CQ) for 2h/4h.Cells were collected to extract cell protein, WB(western blot) detected the autophagosomes formation marker LC3Ⅱ; 5. Autophagy inhibit G-MDSC apoptosis Cultured G-MDSC in RPMI 1640 medium containing TCCM and 3-methyl adenine(3-MA, autophagy inhibitor) for 12h/24 h, the cells were collected, stained with Annexin V/ PI stain, detected the impact of autophagy on G-MDSC apoptosis by FCM. 6. Autophagy increase the function of G-MDSC Cultured G-MDSC in RPMI 1640 medium containing TCCM and 3-MA. Collected the cells for 12 h, and co-cultured the cells with CD4~+ T cell separated from mouse spleen, cultured in 10% FBS RPMI 1640 medium, adding anti-CD3/CD28 antibody to form CD4~+ T cell proliferation system, detected CD4~+ T cell proliferation by 3H-thymidine([3H]-thymidine, 3H-Td R) incorporation assay. Collected the cells for 12h/24 h, stained with DCFDA, then detected the effect of autophagy on the level of cell effector molecule of ROS by FCM; Collected the cells for 12 h, then obtained the total protein, detected the activity of ARG1. 7. G-MDSC autophagy benefit the Lewis transplant tumor development in mice Constructed Lewis transplant tumor in mice, at day 16/23, via the tail vein injection to transferred G-MDSC to mice which had previously blocked the autophagy in vitro in order observe the effect of G-MDSC autophagy on tumor development in mice, as well as cell ratio of IFN-γ expressed cells in the spleen of tumor-bearing mice,including the helper T cell type 1(helper T cell type 1, Th1) and cytotoxic T lymphocytes1(cytotoxic lymphocyte type 1, Tc1) subsets.Results 1. In vitro, different concentrations of TCCM join for G-MDSC culture system to detect its effect on cell viability and effector molecules ROS for 12h/24 h stimulus; the results showed that TCCM favored G-MDSCs survival in vitro, existed concentration-dependent effect; TCCM raised G-MDSCs effector molecule ROS levels. 2. The G-MDSC were cultured in RPMI 1640 system within TCCM and chloroquine(Chloroquine, CQ) for 2h/4h, WB detected the key marker protein molecular LC3Ⅱ of cell autophagsomes formation. The results showed that compared with the control group, after tumor supernatant treatment for 2/4 hours, raised LC3Ⅱlevel, demonstrated under the tumor environment, G-MDSC autophagy level was upregulated(P <0.01) 3. Select TCCM accounting for 1/2 of the volume of culture system to study the effect of autophagy on G-MDSC survival capability, within autophagy inhibitor 3-methyladenine. The results showed that after treated for 24 h, compared with 1/2TCCM group, the proportion of living cells was decresed in the 1/2TCCM+3-MA group,and the results have a statistical difference(P <0.05),indicated that the inhibition of autophagy was conducive for the survival of G-MDSC in vitro. 4. Effect of G-MDSC pre-treatmented in vitro over proliferation capability of CD4~+T cells, the results showed the compared with positive control group(only CD4~+ T in culture system), when G-MDSC exsited in co-culture system, the proliferation of CD4~+ T cells was inhibited in varying degrees. 1/4TCCM+3-MA group compared with 1/4TCCM group, the inhibition capability of G-MDSC on CD4~+ T proliferation was decreased, the results have significant differences(P <0.05). 1/2TCCM+3-MA group compared with 1/2TCCM group, the inhibition capability of G-MDSC was also decreased, the result also has significant difference(P <0.05),indicated that the inhibition of autophagy down-regulated the inhibition capability of G-MDSC on CD4~+ T proliferation in vitro. 5. In vitro culture for 12 h, the cell pellet was acquired and extracted total protein,then detected the arginase activity.Results showed that, 1/2TCCM+3-MA group compared with 1/2TCCM group, when cell autophagy was suppressed, G-MDSC cell arginase activity was decreased, and the results have significant difference(P <0.01). 6. In vitro culture for 12 h, the cells were staining with DCFDA and analyzed by FCM to reflect the ROS activity. The results showed that 1/2TCCM+3-MA group compared with 1/2TCCM group, when cell autophagy was suppressed, ROS activity showed a decreasing tendency. 7. Results showed, at day 22 and 24 after the model have constructed, 1/2TCCM+3-MA group compared with1/2TCCM group, had smaller tumor volume, the results have statistically difference(P <0.05 or P <0.01). The results of tumor weight was consistent with previous results at day 25, and results have significant difference(P <0.01). 1/2TCCM+3-MA group compared with 1/2TCCM group, the proportion IFN-γ+ Tc1 and IFN-γ+ Th1 subsets in spleen became significantly high, and the results have statistical difference(P <0.01),indicated that blocked G-MDSC autophagy decraesed immune suppression function of cells, recoverd the anti-tumor immune response capability of IFN-γ+Tc1 and IFN-γ+ Th1 subsets.Conclusions 1. Tumor cell conditioned medium would raised G-MDSC viability, autophagy, the ability of effector molecules ROS in vitro. 2. In vitro, G-MDSC autophagy can promote cell survival and increase their suppression capability on CD4~+T cell proliferation, and increase the level of its effector molecules ROS and ARG-1. 3. G-MDSC autophagy benefited the development of Lewis transplant tumor in mice, which may be related with the inhibition ability of G-MDSC autophagy on Tc1 and Th1 cells.