A Comparative Study Of Anti-cancer Immune Response Induced By Microvascular Endothelial Cells Vaccine And Tumor Cells Vaccine In Mice Lewis Lung Cancinoma Models

BackgroundLung cancer is one of the most harmful tumors to human health and life,its morbidity and mortality is fast growing in recent years.Recently research has made some progress in stimulating the body's immune system to anti-tumor. Immunotherapy has become an effective new method in the treatment of lung cancer. Tumor vaccines are a kind of immunotherapy to treating the tumor because of tumor associated antigen,which is used to stimulate the immune system to produce anti-tumor immune response.Tumor vaccines play a role in the mechanism of anti-tumor immunotherapy mainly in the expression of tumor associated antigen, particularly tumor specific antigen,can induce the body to produce tumor specific cytotoxic T lymphocytes(CTLs),and then destruct tumor cells.However,tumor vaccines are limited by the fact that tumor cells can escape from immune surveillance not only due to the inherited immunetolerance against their specific antigens,but also through acquired ability to change their specific phenotype and inhibit anti-tumor immunity by decreased expression of HLA and costimulatory molecules,as well as active secretion of immunosuppressive molecules in the tumor microenvironment;In addition,tumor cells of various tissue origins are not only different but also genetically instable;Moreover,immune cells can hardly fully express their cytotoxic potential in the tumor microenvironment.In the 1970s,Folkman proposed that tumor angiogenesis was indispensable for tumor growth and metastasis.In healthy adult tissues,angiogenesis is infrequent due to the balance between inhibitors and stimulators of angiogenesis.When the small tumors are less than 2-3 mm in diameter,the tumor tissue mainly through the role of diffusion to obtain oxygen and nutrients.However,when tumors grow up to the diameter of 2-3 mm,the so called 'angiogenic limit',they can not continue their growth due to insufficient blood supply.Under conditions of hypoxia,tumour cells and interstitial cells like macrophages turn-on the angiogenic switch by secretion of angiogenic stimulators that activate endothelial cells and their precursors to form new blood vessels.As a result,expanding neo-vasculature supports tumours to continue their growth.Tumor vascular endothelial cells vaccines inhibit the process of neovasculature formation or attacks established tumour vessels by specific blocking of tumor nutrition and migration aisle.This immunotherapy has become an important anti-tumor therapeutic strategy.At present,there are more studys about tumor cells vaccines at abroad,while there are only a small number of reports about the study of tumor vascular endothelial cells vaccines.Which anti-tumor effect between the two types vaccines is better remains to be further studied.Experiment aimThis study is to explore mouse brain microvascular endothelial cells(bEnd.3s) vaccine and mouse Lewis lung cancinoma cells(LLCs) vaccine anti-tumor effect and anti-tumor immune response mechanism in mice Lewis lung cancinoma models.Experiment methods1.In vitro experiments:Whole cell lysates of LLCs or bEnd.3s were prepared for freeze-thaw antigens.DCs pulsed with respective freeze-thaw antigen or without freeze-thaw antigen incubated with autogeneic T lymphocytes.T lymphocytes proliferation rate was assessed by CCK-8;Specific kill ratio of CTLs was assessed by MTT.2.In vivo experiments:LLCs and bEnd.3s were prepared for respectively vaccines. After immunized with respectively vaccine,C57BL/6 mice were challenged with subcutaneous injection with LLCs.Tumors growth,mice survival time and adverse effects were observed during the following 90 days.The expression of CD3εand CD8αon spleen T lymphocytes surface from immunized mice were detected by flow cytometry(FCM);CTLs response were assessed by CCK-8; Specific antibodies in serum of immunized mice were detected by ELISA and Western blot.3.All datas were expressed as mean value±S.D and analyzed by statistical software SPSS13.0.P＜0.05 was considered to be statistically significant.Experiment results1.In vitro experiments:Either bEnd.3DCs(DCs pulsed with bEnd.3 freeze-thaw antigen) or LLC2.DCs(DCs pulsed with LLC freeze-thaw antigen) showed a greater ability to stimulate T lymphocyte proliferation than DCs(DCs pulsed without freeze-thaw antigen) and Ts(T lymphocytes incubated solitary)(P＜0.05).Compared with the respective control group,CTLs from either bEnd.3DCs or LLCDCs showed a higher specific kill ratio against the respective target cells when the ratio of effector to target was 30:1(P＜0.05).3.In vivo experiments:(1) Tumor volumes of bEnd.3s-immunized mice and LLCs-immunized mice were smaller than PBS control group,especially tumor volumes of bEnd.3s-immunized mice were the smallest,and the differences among ench group were statistically significant(P＜0.05);The histopathological examination showed that the tumor tissue was not found in bearing cancer parts of bEnd.3s-immunized mice, whereas found in bearing cancer parts of LLCs-immunized mice and PBS control group;BEnd.3s-immunized mice could survival with tumor-free in a long-term, while the median survival time of LLCs-immunized mice and PBS control group were 81 days and 44 days respectively;Compared with LLCs-immunized mice and PBS control group,the intention time of bEnd.3s-immunized mice was slower.(2) The expression rate of CD3εand CD8αon spleen T lymphocytes surface was 18.7%in bEnd.3s-immunized mice,14.7%in LLCs-immunized mice,and 10.7% in PBS control group,and the differences among ench group were statistically significant(P＜0.05).(3) CTLs from either bEnd.3s-immunized mice or LLCs-immunized mice but not PBS control group,showed a strong lytic activity against the respective target cells.Specific CTLs from bEnd.3s-immunized mice targeting against bEnd.3s; Specific CTLs from LLCs-immunized targeting against LLCs;Compared with respective conrtol group,the differences were statistically significant(P＜0.05).(4) The anti-serum antibody titer of bEnd.3s-immunized mice was 1:1600,while the anti-serum antibody titer of LLCs-immunized mice was 1:800.There was a reactivity of antibodies in bEnd.3s-immunized mice serum with bEnd.3 membrane proteins,whereas not with LLC membrane proteins;There was a reactivity of antibodies in LLCs-inmuzed mice serum with LLC membrane proteins,whereas not with bEnd.3 membrane proteins.(5) There were some positive reaction zones from bEnd.3s-immunized mice serum reaction with bEnd.3 membrane proteins,of which 220KD was consistent with the molecular weight of vascular endothelial growth factor receptor-2(VEGFR-2) and 180KD was consistent with the molecular weight of Endoglin.There were some positive reaction zones from LLCs-immunized mice serum reaction with LLC membrane proteins,of which 200KD was consistent with the molecular weight of carcino-embryonic antigen(CEA).Experiment conclusion1.Microvascular endothelial cells vaccine and tumor cells vaccine could induce specific cellular and humoral immunity,and as a consequence inhibited Lewis lung cancinoma subcutaneous transplantation tumor growth and prolonged mice ??survival time.2.Microvascular endothelial cells vaccine showed better effect on mice Lewis lung cancinoma models in comparison with tumor cells vaccine.