A Immunity Response On Anti-angiogenesis Of Lung Cancer Induced By Allogenic Mouse Brain Micro-vascular Endothelial Cell Vaccine

Worldwide,lung cancer is the most common cancer in terms of both its incidence and mortality with the highest rates.The incidence of lung cancer went up to 111.85%from the 1970s to the 1990s in China.The present treatment modes of lung cancer,mainly including surgery and supplemental chemotherapy and radiation, couldn't decrease the mortality rates and its metastasis rates currently.It is necessary for us to seek more rational and effective strategy to treat lung cancer.With the continuous development of immunology,molecular biology and genetic engineering technology,immunotherapy has emerged as the fourth selection for cancer therapy. Tumor biotherapy,as a representative of the immunotherapy,has been evoked great interest now.Tumor biotherapy mainly transfers or enhances self anti-tumor ability to suppress tumor growth,which can avoid the serious shortcomings of currently tumor therapy modes.It shows a good clinical application prospect.Now,most of the drugs for cancer molecular targeting therapy have been developed successfully and listed,such as Iressa,Sutent,Tarceva,Veenat and Nexavar Sorafenib.These drugs mostly are monoclonal antibodies and are targeted to the specific protein molecules of tumor-derived vascular endothelial cells.To some extent, these drugs can suppress the tumor growth and prolong the patient survival,but their therapeutic effect still unsatisfactory from some evidence-based research studies. Additionally,monoclonal antibodies can result in serious adverse effects and too expensive to afford for most patients if being used for a long term.All of these disadvantages have limited their clinic application.Active immunotherapy of targeting tumor vascular endothelial cells plays an important role in tumor biotherapy.Its therapeutic effects can maintain a long time and less adverse effect.Furthermore,it needn't repeat dosing frequently and can induce humoral immune or cellular immune response.Anti-angiogenic therapy,which targets tumor vascular endothelial cells,might represent an attractive strategy for the treatment of tumors.Tumor vascular endothelial cells are actively proliferative and there are many specific protein molecular expressing on their membrane surface, which is correlating with cell proliferation,such as vascular endothelial growth factor receptorⅡ(VEGFR-Ⅱ),integrin avβ3,Endoglin and tissue factor,etc.But,they can't be detected on membrane surface of dormancy endothelial cells in vivo.Many researches have indicated that it can successfully break immune tolerance and induce specific immune response of anti-angiogenesis by using the proliferation activity xenogeneic endothelial ceils as vaccine in vitro.Recently,some studies on the vaccine prepared from proliferation related proteins and DNA molecular of xenogeneic or allogenic tumor vascular endothelial cells have been reported and made some progress.But it has received much concern on its adverse effects,such as hypersensitivity and their single therapy target,etc.Therefore,to decrease side effects and increase multi-targets,it is imperative to discover a new and rational immunotherapy method.In the present study,proliferating micro-vascular endothelial cells(bEnd.3) derived from mouse brain micro-vascular were cultured in vitro.We immunized the mice with a vaccine of glutaraldehyde-fixed bEnd.3 in a transplanted Lewis lung cancer model and in a lung metastasis model of cervical cancer U14.At the same time, we immunized the mice with DCs loading bEnd.3 antigen in a transplanted Lewis lung cancer model.The anti-tumor immune response induced by these different vaccines was evaluated and the mechanism of the anti-tumor effects was discussed preliminarily.The aim of the study was to break tolerance and elicited active immune response against tumor endothelial cells,in order to explore a new anti-angiogenesis approach for cancer immunotherapy.It was reported that human umbilical vein endothelial cells(HUVEC) derived from marco-vascular could induce the anti-angiogenesis effect.Therefore,in the whole experimental process,HUVEC were used as a positive control and mouse fibroblasts(NIH3T3) were used as a negative control to be observed the anti-angiogenesis effect in mice.PartⅠAnti-angiogenesis immunity response of lung cancer induced by aliogenic bEnd.3 cells vaccineChapter 1 Culture of bEnd.3 and preparation of vaccinesMethods:1.Proliferating allogenic bEnd.3 and xenogeneic HUVEC were cultured in vitro. vWF,CD31 and CD144 which were the surface markers of endothelial cells were detected through immunohistochemical and RT-PCR methods.2.The expression of VEGFR-Ⅱand integrin av were detected by RT-PCR method in bEnd.3,HUVEC,NIH3T3 and tumor cells(LLCs and U14).3.The bEnd.3,HUVEC,NIH3T3 cells were proliferated massively and fixed by 0.025%glutaraldehyde and prepared to vaccines.The vaccine's concentration was 2.5×10~7/ml.Results:1.Proliferating allogenic bEnd.3 and xenogeneic HUVEC were cultured successfully and were proliferated massively in vitro.The two cells have expressed highly vWF,CD31 and vWF,CD144,respectively.2.In mRNA level,bEnd.3 and HUVEC expressed VEGFR-Ⅱgene and Integrin av gene highly,but didn't express in NIH3T3.LLCs and U14 expressed highly Integrin av gene,but didn't express VEGFR-Ⅱgene.3.Vaccines could prepare successfully by 0.025%glutaraldehyde.Chapter 2 Detection of anti-tumor effect induced by allogenic bEnd.3 vaccine to a transplanted Lewis lung cancer model Methods:1.Two treatment protocols in a lung transplanted Lewis lung cancer model i.e., preventive and therapeutic ones were used.In the preventive protocol,vaccines were divided into four treatment groups:control(PBS only),bEnd.3(allogenic endothelial vaccine),HUVEC(xenogeneic endothelial vaccine),and NIH3T3 (allogenic fibroblast vaccine) groups(n=15 each),which were given weekly subcutaneous injections of the respective vaccines at the axillary lymph node (5×10~6 fixed cells/0.2 ml/mouse) for 5 consecutive weeks.One week after the last vaccination,a single cell suspension of LLC cells in Hanks' Balanced Salt Solution was injected subcutaneously(3×10~6 cells/0.2 ml/mouse).In the therapeutic protocol(n=6 each),vaccines were divided into four treatment groups same as the preventive protocol.Mice were divided into serum and T lymphocytes groups derived from the preventive immunized mice and detected positive by ELISA were used to inject into the mice bearing Lewis lung cancer intramuscularly and subcutaneously every other day for 6 times,respectively. Injection dose of serum and T lymphocytes were 50μl/mouse and 1×10~7 cells/0.2 ml/mouse.2.Tumor volume and mice survival were observed after 6 weeks.The changes of tumor tissue were detected by staining with HE.The specific CTLs cytotoxicity activities of spleen T lymphocytes derived from the immunized mice examined by CCK assay.The ratio of CD3~+CD8~+ T lymphocytes was measured by FACS. Antibody specificity was detected by ELISA and immunohistochemical methods. The effect on anti-sera derived from the immunized mice to proliferation of target cells was examined by CCK assay also.At last,activities of anti-VEGFR-Ⅱantibody and anti-integrin av antibody in anti-sera were detected with Western blot.3.Effect of vaccination on wound healing:a cross wounds of 7-mm diameter each were inflicted on the upper back of C57BL/6 mice 2 week after the last vaccination.Effect of vaccination on wound healing was observed. Results:1.The tumor growth in a transplanted Lewis lung cancer model was surpressed and survival of mice was prolonged.In the preventive group and therapeutic group,endothelial vaccination induced a significant inhibition of growth of transplanted Lewis lung cancer in mice(P＜0.05).The tumor volume of 100%of mice immunized with bEnd.3 vaccine and 90%of mice immunized with HUVEC decreased to 20 mm~3 3 weeks after the challenge with tumor cells and arrived tumor-free at the end of test(90 days).Compared with control,it was significant statistically(P＜0.05).The median survival time of bEnd.3 group and HUVEC group were 90 days,which was more prolonged significantly than controls(P＜0.05).Tumors grew slower in mice of T lymphocytes and serum treatment groups than controls(P＜0.05).But the mouse survival of T lymphocytes treatment groups could be prolonged more than serum treatment groups(P＜0.05).2.The specific cytotoxic T iymphocytes immune response targeting to tumor vascular endothelial cells were induced.To confirm the functional activity and specificity of the cellular immunity activated by endothelial vaccination,spleen T lymphocytes isolated from immunized mice used as effectors in CCK assay against endothelial or tumor targets.BEnd.3-induced CTLs at a 25:1 ratio showed a significant lytic activity against bEnd.3 when compared to the CTLs of control mice.HUVEC-induced CTLs showed same lytic activity to HUVEC.However,immune effects of both groups showed no significant lytic activity against LLC cells when tested under the same conditions.The ratio of CD3~+CD8~+ spleen T lymphocytes in bEnd.3 immunized mice was higher than controls(P＜0.05).3.The specific antibody immune response in anti-sera derived from mice immunized was induced.Antibody specificity detected by ELISA and immunohistochemical methods showed the anti-sera of mice with endothelial cells had specific immune response with endothelial cells,but not tumor cells.The anti-sera could also inhibit the proliferation of endothelial cells in vitro.Western blot results showed that anti-sera of mice receiving bEnd.3 or HUVEC vaccinations precipitated membrane proteins of the respective endothelial cells,which both appeared as specific bands of approximately 220 KD and 180 KD and didn't appear as specific bands of approximately 130 KD in bEnd.3 but in HUVEC.The specific bands couldn't see in target LLC cells.4.Wound healing time was delayed.Wound healing time was delayed in bEnd.3 and HUVEC groups compared with NIH3T3 group and PBS group(P＜0.05).Chapter 3 Detection of anti-tumor effect induced by allogenic bEnd.3 vaccine to a lung metastasis model of cervical cancer U14Methods:1.Two treatment protocols in a lung metastasis model of cervical cancer U14 i.e., preventive and therapeutic ones were used.In the preventive protocol,vaccines were divided into four treatment groups(n=10 each),control(PBS only),bEnd.3 (allogenic endothelial vaccine),HUVEC(xenogeneic endothelial vaccine),and NIH3T3(allogenic fibroblast vaccine) groups,which were given weekly subcutaneous injections of the respective vaccines at the axillary lymph node (5×10~6 fixed cells/0.2 ml/mouse) for 5 consecutive weeks.One week after the last vaccination,a single cell suspension of cervical cancer U14 cells in Hanks' Balanced Salt Solution was injected into the tail veins of mice(5×10~6 cells/0.2ml /mouse).In the therapeutic protocol,vaccines were divided into four treatment groups same as the preventive protocol,which were injected with tumor cells (day 0) prior to vaccination on days 1,3,5,7,9,and 11.2.Lung metastasis was evaluated macroscopically by counting metastatic nodules that were clearly visible on the lung surface.Mice survival was observed after 17 day.The changes of tissue were detected by staining with HE.The specific CTLs cytotoxicity activities of spleen T lymphocytes derived from the immunized mice examined by CCK assay.The ratio of CD3~+CD8~+ T lymphocytes was measured by FACS.Antibody specificity was detected by ELISA and immunohistochemical methods.The effect on anti-sera derived from the immunized mice to proliferation of target cells was examined by CCK assay also.At last,activities of anti-VEGFR-Ⅱantibody and anti-integrin av antibody in sera were detected with Western blot.Results:1.Lung metastasis of cervical cancer U14 was inhibited and survival of mice was prolonged.In lung metastasis model of cervical cancer U14 experiment,metastatic nodules of left lung surface in bEnd.3 and HUVEC group were less than controls significantly(P＜0.05),especially in preventive group.The median survival time of bend.3 group was 34 days in preventive group and 26 days in therapeutic group,whereas 19 days in negative control.It showed significant statistically(P＜0.05).2.The specific cytotoxic T lymphocytes immune response targeting to tumor vascular endothelial cells were induced.In CFSE and PI assay,bEnd.3-induced CTLs and HUVEC-induced CTLs at a 30:1 ratio showed a significant lyric activity against bEnd.3 and HUVEC when compared to the CTLs of control mice(P＜0.05).However,immune effects of both groups showed no significant lyric activity against U14 cells when tested under the same conditions.The ratio of CD3~+CD8~+ spleen T lymphocytes in bEnd.3 immunized mice was higher than negative controls(P＜0.05).3.The specific antibody immune response in anti-sera derived from mice immunized was induced.Antibody specificity detected by ELISA and immunohistochemical method showed the anti-sera of mice immunized with endothelial cells had specific immune response with endothelial cells,but not tumor cells.Furthermore,the anti-sera of mice immunized with U14 cells had immune response with bEnd.3. The anti-sera of mice immunized with endothelial cells also could inhibit the proliferation of endothelial cells in vitro.Western blot results showed that anti-sera of mice receiving bEnd.3 or HUVEC vaccinations precipitated membrane proteins of the respective endothelial cells,which appeared as specific bands of approximately 220 KD and 180 KD in bEnd.3 and approximately 220 KD and 130 KD in HUVEC.The specific bands couldn't see in target U14 cell s.PartⅡAnti-angiogenesis immunity response of lung cancer induced by DCs loading allogenic bEnd.3 cells antigenChapter 1 Preparation of bEnd.3 antigen and culture of DCsMethods:1.Whole cell lysates of bEnd.3,HUVEC or NIH3T3 were prepared by four rapid freeze-thaw cycles.2.DCs were cultured from mice bone marrow in vitro and identified by morphology and surface markers i.e.,CD11a and CD86 by FACS.3.The antigen concentration of loading DCs was 100μg/ml.Results:1.It proved that the cells were lysed completely after freeze-thaw method by trypan blue staining.Quantitative detection of antigen showed that 1×10~7 cells could get 398.98±96.36μg antigen.2.DCs were cultured successfully from mice bone marrow in vitro and were mature at day 8.Chapter 2 Anti-angiogenesis response induced by DCs loading bEnd.3 antigen in vitroMethods:1.Freshly isolated DCs were incubated with the respective cell lysate for 72 h at a final protein concentration of 100μg/ml.Spleen T lymphocytes isolated from non-immunized mice mixed with DCs loading antigens for 3 days at 20:1 and 40:1.Spleen T lymphocytes proliferation in vitro was observed.2.CTLs induced by DCs loading antigens mixed with target cells for 3 days and cytotoxic activity were detected by MTT assay.Results:1.Proliferation of Spleen T lymphocytes was promoted in vitro.Spleen T lymphocytes proliferation assay showed that DCs loading antigens of bEnd.3,HUVEC and NIH3T3 antigen could make the T lymphocytes proliferate in vitro(P＜0.05) at 20:1 and 40:1 compared with DCs group and PBS group.2.The specific cytotoxic T iymphocytes immune response targeting to tumor vascular endothelial cells were induced in vitro.The cytotoxic activity detected by MTT assay showed that bEnd.3DCs-induced CTLs and HUVECDCs-induced CTLs at a 30:1 ratio had a significant lytic activity against bEnd.3 and HUVEC when compared to the NIH3T3DCs-induced CTLs group,DCs group and PBS group mice(P＜0.05). However,immune effects of both groups showed no significant lytic activity against LLC cells when tested under the same conditions(P＞0.05).Chapter 3 Anti-angiogenesis in a lung transplanted Lewis lung cancer model induced by DCs loading bEnd.3 antigen in vivoMethods:1.DCs loading antigens were collected at day 8 as vaccine.Mice were divided into five treatment groups:bEnd.3DCs,HUVECDCs,NIH3T3DCs,DCs and PBS group and given weekly subcutaneous injections of the respective vaccines at the axillary lymph node(1×10~6 fixed cells/0.2 ml/mouse) for 5 consecutive weeks. One week after the last vaccination,a single cell suspension of LLC ceils in Hanks' Balanced Salt Solution was injected subcutaneously(3×10~6 cells/0.2 ml /mouse).2.Tumor volume and mice survival were observed after 6 weeks.The changes of tissue were detected by staining with HE.The specific CTLs activities of spleen T lymphocytes derived from the immunized mice examined by CCK assay.The ratio of CD3~+CD8~+ T lymphocytes was measured by FACS.Antibody specificity was detected by ELISA and immunohistochemical methods.At last,activities of anti-VEGFR-Ⅱantibody and anti-integrin av antibody in anti-sera derived from the immunized mice were detected with Western blot.Results:1.The tumor growth in a transplanted Lewis lung cancer model was surpressed and survival of mice was prolonged in vivo.3 weeks after incubation with LLCs,tumors grew slower in mice immunized with bEnd.3DCs and HUVECDCs vaccines than NIH3T3DCs groups, DCs groups and PBS groups(P＜0.05).But the tumors of NIH3T3DCs group and DCs group grew slower than PBS groups'(P＜0.05).The median survival time of bEnd.3DCs group,NIH3T3DCs group and DCs group was 65 days,57 days and 55 days,whereas 19 days in PBS group,which showed significant statistically(P＜0.05).2.The specific cytotoxic T lymphocytes immune response targeting to tumor vascular endothelial cells were induced in vivo.The cytotoxic activity detected by CCK assay showed that bEnd.3DCs-induced CTLs and HUVECDCs-induced CTLs at a 30:1 ratio had a significant lytic activity against bEnd.3 and HUVEC when compared to the CTLs of NIH3T3DCs group,DCs group and PBS group mice(P＜0.05).The ratio of CD3~+CD8~+ spleen T lymphocytes from bEnd.3 immunized mice was higher than negative controls(P＜0.05).3.The specific antibody immune response in anti-sera derived from mice immunized was induced in vivo.Antibody specificity detected by ELISA indicated that antibody specificity to endothelial cells had no marked difference before and after the incubation with LLC cells.But antibody specificity in the serum,excluded PBS group could make response with endothelial cells and LLC cells.It suggest the important function of DCs.Western blot results showed that anti-sera of mice receiving bEnd.3DCs or HUVECDCs vaccinations precipitated membrane proteins of the respective endothelial cells,which both appeared as specific bands of approximately 220 KD and 180 KD and didn't appeare as specific bands of approximately 130 KD in bEnd.3DCs but in HUVECDCs.The specific bands couldn't see in target LLC cells.Conclusions1.Allogenic bEnd.3 vaccine and DCs vaccine loading allogenic bEnd.3 antigen can surpress the tumor growth and metastasis in a transplanted Lewis lung cancer model and a lung metastasis model of cervical cancer U14 and prolong survival of mice.This suppressive effects is more effective in preventive groups than therapeutic group.2.The anti-tumor effect is attributed to obtain endothelial cell specific CTLs and induce production of antibodies that can react with VEGFR-Ⅱ,intergrin av or endoglin proteins etc expressing on tumor endothelial cells in mice.The endothelial vaccine induces an active specific cellular and humoral immune to anti-angiogenesis.3.Although the endothelial vaccines can delay the wound healing time,the wound can heal completely at last.4.Allogenic and xenogeneic vaccines can both induce anti-angiogenesis active immune response.5.Compare DC vaccine loading endothelial cells antigen with the whole endothelial cell vaccine,the latter has more effective anti-tumor effect than the former.It indicates that the whole endothelial cell vaccine can yet be regarded as a promising research direction of anti-angiogenesis.