A Study On The Changes Of Immune Function During Peri-operative Period In Lung Cancer Patients And Its Correlation With Tumor Metastasis

Objectives:To investigate the changes of cell-mediated immunity,myeloid-derived suppressor cells(MDSCs)and its subpopulations and regulatory T cells(Treg)during peri-operative period in lung cancer patients.To evaluate the influence of surgery-induced MDSCs on tumor growth and angiogenesis in lung cancer patients.To investigate the correlation of surgery-induced MDSCs with tumor metastasis and the influence of bone marrow mesenchymal stem cells(BMSCs)on the induction of MDSCs and the development of post-operative tumor metastasis in mouse tumor model.To evaluate the correlation between MDSCs,monocytic MDSCs(M-MDSCs),granulocytic MDSCs(G-MDSCs)and Treg in peri-operative period in lung cancer patients.Methods:The concentration and percentage of lymphocytes and its subpopulations,CD11lb+CD33+HLA-DR-MDSCs and M-MDSCs,G-MDSCs,Treg cells in peripheral blood mononuclear cells(PBMCs)were determined in pre-,intro-and post-operative period,specifically 24 hours,3 and 7 days after surgery(depicted as T1,T2,T3,T4 and T5 respectively)by blood cell analyses and flow cytometry(FCM)in 111 lung cancer patients.The concentration of VEGF and Thl/Th2 cytokines in serum was also detected using enzyme-linked immunosorbent assay and cytomic bead array techniques.To observe the influence of CD11b+CD33?HLA-DR-cells on tumor growth and angiogenesis,human lung cancer A549 cells combined with peri-operative isolated CD11b+CD33+HLA-DR-cells were injected subcutaneously into nude mice.LLC cells were injected intravenously into C57BL/6 mice.Two hours later,these mice were divided into 4 groups:controls(C group);mice given anesthesia(A group);mice given anesthesia and laparotomy(AL group)and mice given anesthesia,laparotomy,and hepatic lobectomy(ALH group).The AL mice were further divided into 2 groups after surgical operation:the AL mice without treatment and the AL mice treated with syngeneic BMSCs(ALB group).The percentage of Gr-1+CD11b+ cells in PBMCs was detected by FCM.The numbers of metastases on the lung surface were counted on day 14 after LLC infusion.BMSCs were also co-cultured in vitro with myeloid cells in order to illustrate the effects of BMSCs on the generation of MDSCs.The correlation between MDSCs and Treg was further analyzed at T1-T5.M-MDSCs,G-MDSCs and CD4+ cells isolated using FCM cell sorting and anti-CD4 magnetic beads were cocultured.Five days after coculture,Treg cells were analyzed by FCM.Results:The concentration of CD3+ T cells and NK cells decreased significantly in post-operative period compared with pre-operative period(P<0.05).There was no statistic significant when the concentration of NKT cells and the ratio of CD4/CD8 in post-operative period were compared with pre-operative period(P>0.05).Compared with the pre-operative period,the concentration of IL-5 and IL-10 in serum was increased in post-operative period(P<0.05).Either the percentage or the concentration of MDSCs and M-MDSCs significantly increased in lung cancer patients after surgery as compared with pre-operation(P<0.01).The concentration of Treg cells significantly increased at T5 as compared with T1(P<0.05).Compared with the pre-operation,the concentration of pro-angiogenic cytokine VEGF in serum was increased in post-operative period.Isolated MDSCs after surgery were more efficient in promoting angiogenesis and tumor growth than MDSCs isolated before surgical operation in allograft tumor model.The numbers of lung metastasis in AL and ALH group significantly increased as compared with C group(P<0.05)and the numbers of lung metastasis in ALH group significantly increased as compared with A group(P<0.05).The percentage of Gr-1?CD11b+ cells in PBMCs at post-operative period significantly increased in A,AL and ALH group as compared with C group(P<0.05).The numbers of lung metastasis in AL and ALB group were(15.38:± 1.12)and(4.38±0.73)(P<0.01)on day 14 after LLC infusion.Meanwhile,the percentage of Gr-1+CD11b+ cells in ALB group significantly decreased as compared with AL group(P<0.01).We also demonstrated that BMSCs inhibited the induction and proliferation of MDSCs from myeloid cells in vitro.There was a positive correlation between MDSCs and Treg at T3-T5(P<0.05).The number of Treg cells induced by MDSCs isolated at T3-T5 was increased as compared with T1(P<0.01).Conclusions:There is suppression in the cellular immunity of lung cancer patients in post-operative period.The Th1/Th2 balance tilts towards Th2.Post-operatively induced MDSCs and M-MDSCs were significantly increased and qualified with potent pro-angiogenic and tumor promotive ability.Surgery stress induces MDSCs and promotes tumor metastasis,and syngeneic BMSCs inhibit the generation of MDSCs and further suppress tumor metastasis after surgery.The surgery-induced M-MDSCs induce proliferation of Treg.