Anti-tumor Efficacy Of Combined Vaccine Based On The Induced HUVEC And DC-CT26 Against Mouse Colorectal Carcinoma

The growth and metastasis of solid tumors depend on neovascularization.Therefore,inhibition of tumor progression through anti-angiogenesis has become a hot topic for medical researchers.Recent studies have demonstrated that vaccines taking blood vessel related antigen as a target can make the body to produce specific cellular and humoral immune responses and then prevent tumor growth and metastasis by inhibiting neovascularization.Among them,antiangiogenic study based on whole-cell vaccines prepared by taking the human umbilical vein endothelial cell(HUVEC)antigens showed that although the vaccine can cause a certain degree of immune response,the effect is still limited.The vaccine needs further optimization.There are significant differences between tumor vascular endothelial cells and normal vascular endothelial cells in various aspects,such as the growth factor receptors,signal transduction molecules and adhesion molecules.We hypothesized that simulating tumor microenvironment in vitro to induce HUVEC can make it close to the properties of tumor vascular endothelial cells.Applying induced HUVEC as a vaccine might have a better suppressive effect on tumor angiogenesis than HUVEC vaccine.After consulting the literature,no research has been reported in this area.In the previous study of our lab,the method of simulating tumor microenvironment with tumor cell supernatant has been established in vitro.In this study,we used the supernatant of murine colorectal carcinoma CT26 cells to simulate tumor microenvironment to induce HUVEC.They all had high expression of tumor vascular endothelial cell markers,enhanced capabilities of migration and invasion,which was similar to tumor vascular endothelial cells.We call induced HUVEC"tumorous HUVEC".The body has immune tolerance to its own or allogeneic antigens,but heterologous vaccines can break the body's immune tolerance to autoantigen.It can be easily recognized and rejected by the host's immune system.Inoculation of mice with HUVEC induced by tumor microenvironment can destroy the immune tolerance and induce specific antiangiogenic immune responses,then destroy tumor neovascularization,and inhibit tumor growth.Dendritic cells(DC)are the most powerful professional antigen presenting cells in the body.They can take up tumor antigens and induce CTL-mediated strong specific anti-tumor immune effects.At present,there are various kinds of DC vaccines.In this study,DC vaccine was adopted to load freeze-thaw antigens from CT26 cells in mice.It was DC-CT26 vaccine.The vaccine carries all tumor antigens without the need for isolation of specific antigen peptides,without determining the haplotypes of MHC and MHC-restricted antigenic determinants,and induces immune responses of targeting polyclonal cytotoxic T-lymphocytes(CTL),and then produces anti-tumor effects.Colorectal carcinoma(CRC)is one of the most common deadliest cancers worldwide with its incidence on the rise.More than 1.2 million new cases are diagnosed and more than 600,000 people die from the disease every year.Data from the National Cancer Center in 2017 showed that it had the fifth highest incidence and mortality in China.Early colorectal carcinoma is asymptomatic.Therefore,this type of malignant tumor is usually diagnosed late.The 5-year survival rate of colorectal carcinoma is less than 10%.Therefore,there is an urgent need to develop new strategies for the treatment of colorectal carcinoma.In this study,murine colorectal carcinoma cell supernatant was used to induce HUVEC to prepare induced HUVEC vaccine for developing anti-tumor effect mainly through humoral immunity."DC-CT26"vaccine which was loaded with CT26 cell freezing and thawing antigens could kill colorectal carcinoma cells mainly through cellular immunity.On the one hand,the combination of two vaccines could produce a suppressive effect on colorectal vascular endothelial cells,and on the other hand,it could produce a suppressive effect on colorectal carcinoma cells,and then the anti-tumor effect could be improved.Part I the Effect of Induced HUVEC Vaccine on Anti-mouseColorectal Tumor AngiogenesisMaterials and Methods1.Tumor microenvironment was simulated with murine colorectal carcinoma CT26 cell supernatant.The effect of tumor microenvironment on HUVEC was detected by wound-healing assay,invasion assay and western blot.2.Induced HUVEC vaccine:(1)Collect the supernatant of CT26 cells:CT26cells were routinely cultured.When they reached about 80%confluence,5ml/dish of1640 medium containing 10%fetal calf serum was replaced.After 24 hours,the culture solution was collected into a centrifuge tube and centrifuged at 3000r/min for5 minutes.The supernatant was collected into a new centrifuge tube and stored at-20°C for use.(2)Preparation of induced HUVEC vaccine:HUVEC were routinely cultured.When the cells grew to 60%confluence,the conditioned medium was changed.The amount of the tumor conditioned medium was 10ml/dish.After cultured for 48 hours,induced HUVEC was obtained.Cells were digested,centrifuged,and the supernatant was discarded.0.025%glutaraldehyde was added to the cells.After the mixture,the cells were fixed at 4?and protected from light for 20 minutes.Resuspension concentration in PBS was 2.5×10~7/ml,then the vaccine was stored at-80?for use.3.Experimental animals and grouping:21(4 to 6 weeks old)female BALB/c mice of SPF grade(about 20-22g)were reared in a barrier system and randomly divided into the PBS group,the HUVEC group and the induced HUVEC group.Injection of the corresponding vaccines around the axillary lymph node in mice:The PBS group,0.2ml PBS as a blank control,once a week for 5 weeks.The HUVEC group,0.2ml prepared HUVEC vaccine(2.5×10~7/ml)was injected once a week for 5weeks.The induced HUVEC group,0.2ml prepared induced HUVEC vaccine(2.5×10~7/ml)was injected once a week for 5 weeks.On the 7th day after the last inoculation,CT26 cells(1×10~5cells/body)were injected subcutaneously into the back of the mice in each group.On the eleventh day after CT26 cells were inoculated,the tumors were measured every other day and the tumor growth and survival of the mice were observed.On the 24th day after CT26 cells were inoculated,the mice were sacrificed by pulling their necks after the blood collection from the eye socket.4.Management of tumor specimens:the tumor tissues were collected and photographed.Some of the tumor tissues were fixed with 10%neutral formaldehyde,embedded in paraffin and routinely made for immunohistochemistry.The others were stored at-80°C for hemoglobin assay and western blot assay.5.The expression of CD31 in tumor tissues was detected by immunohisto-chemistry to determine the angiogenesis of the tumor tissues.Hemoglobin assay detected the concentration of hemoglobin in the supernatant of the tumor tissues and indirectly reflected the angiogenesis of the tumor tissues.Western blot was used to detect the expression levels of tumor vascular endothelial cell markers(TEM1,TEM8and VEGFR2)to determine angiogenesis in the tumor tissues.6.The mice serum of each group was extracted and applied as a primary antibody to incubate with the total protein of induced HUVEC.Western blot was applied to test the expression levels of proliferation related antibodies.ELISA assay was used to detect the antibody level of anti-HUVEC in the mice serum of each group.Result1.Wound-healing test showed that the 60%of the CT26 cell supernatant group had the highest number of migratory endothelial cells compared with the PBS group and the 40%of the CT26 cell supernatant group(P<0.001,respectively).2.Invasion assay showed that the 60%of the CT26 cell supernatant group had the highest number of invasive endothelial cells compared with the PBS group and the 40%of the CT26 cell supernatant group(P<0.001,respectively).3.Western blot showed that the expression levels of TEM1,TEM8 and VEGFR2,the specific markers of tumor endothelial cells were significantly increased in the60%of the CT26 cell supernatant group compared with the PBS group(P<0.001).4.The tumor volume and weight results showed that the mice in the induced HUVEC group had the smallest tumor volume(P<0.001,P<0.05)and the lightest weight(P<0.001,P<0.05)compared with the PBS group and the HUVEC group.5.Immunohistochemistry results showed that the density of microvessels in the induced HUVEC group was the lowest in the tumor tissues compared with the PBS group and the HUVEC group(P<0.001,P<0.01).The hemoglobin test results showed that the hemoglobin content of the induced HUVEC group was the least compared with the PBS group and the HUVEC group(P<0.001,P<0.01).Western blot results in tumor tissues showed that the tumor angiogenesis was the least in the induced HUVEC group compared with the PBS group and the HUVEC group(P<0.001,P<0.01).6.Positive bands were detected in the mice serum of each group at the levels of165 kD,130 kD,and 63 kD by western blot,similarly with the bands produced by the incubation of TEM1,VEGFR2,and TEM8 antibodies with the total protein of induced HUVEC.It was presumed that the positive bands might be TEM1,VEGFR2,and TEM8.7.The results of ELISA showed that the HUVEC antibody produced in the induced HUVEC group had the highest titer compared with the PBS group and the HUVEC group(P<0.001,P<0.01).Part II Induced HUVEC Vaccine Combined with DC-CT26Vaccine against Mouse Colorectal CarcinomaMaterials and Methods1.Preparation of DC-CT26 vaccine:CT26 cell antigens were obtained by repeated freezing and thawing.The concentration of CT26 cell lysate was measured and stored at-80°C for use.On day 5 of the culture of murine bone marrow-derived DC,CT26 cell lysate(100?g/ml)was added and then cultured to the 8th day.After DC were digested by trypsin,DC-CT26 vaccine was obtained.And then it was resuspended with PBS to adjust the concentration to 5×10~6/ml.2.Experimental animals and grouping:140(4 to 6 weeks old)female BALB/c mice of SPF grade(about 20-22g)were reared in a barrier system and randomly divided into the PBS group,the DC-CT26 group,the induced HUVEC group and the combined group.Eight mice in each group were injected with the corresponding vaccines around the axillary lymph nodes of the mice.The PBS group,each was injected with 0.2ml of PBS as a blank control once a week for 5 weeks.The DC-CT26 group,each was injected with 0.2ml DC-CT26 vaccine(5×10~6/ml)once a week for 5 weeks.The induced HUVEC group,each was injected with 0.2ml induced HUVEC vaccine(2.5×10~7/ml)once a week for 5 weeks.The combined group,0.2ml DC-CT26 vaccine was injected around one side of axillary lymph nodes(5×10~6/ml),and 0.2ml induced HUVEC vaccine(2.5×10~7/ml)was injected around the other side of the axillary lymph nodes once a week for 5 weeks.3.From the 10th day with the same method,the tumors were measured every other day and the growth of the tumor and the survival of the mice were observed.On the 28th day after the CT26 cells were injected,all the mice were sacrificed after taking the blood from the eye socket.All the tumor tissues were collected and weighed and photographed.The immunohistochemistry,hemoglobin assay,Western blot,and ELISA assay were also used to detect neovascularization in the tumor tissues and endothelial antibodies produced by these mice.4.Peel the spleen tissues of mice in each group,then weigh and photograph the spleen tissues.The spleen index was calculated to examine the immune function of the body.The killing effect of CTL in mice on CT26 cells was detected with the instructions of CytoTex96~?non-radioactive cytotoxicity test kit.The level of IFN-?in mouse serum was tested in accordance with the mouse IFN-?enzyme linked immunosorbent assay kit.Lymphocytes were isolated from the spleen of mice.The expression levels of CD3~+and CD8~+T lymphocytes in the spleen of each group were detected by flow cytometry.Result1.The tumor volume and weight of the mice in each group showed that the tumor volume(P<0.05,P<0.01,P<0.01)and weight(P<0.05,P<0.001,P<0.001)of the DC-CT26 group,the induced HUVEC group and the combined group were significantly decreased compared with the PBS group.The tumor volume(P<0.01,P<0.05)and weight(P<0.05,respectively)of the combined group were significantly decreased compared with the DC-CT26 group and the induced HUVEC group.Tumor inhibition rate results showed that the combined group had the highest inhibition rate compared with the DC-CT26 group and the induced HUVEC group(P<0.001,P<0.05).2.Immunohistochemical results showed that the density of microvessels in the induced HUVEC group and the combined group were obviously decreased compared with the DC-CT26 group and the induced HUVEC group(P<0.001,respectively).The combined group had the least microvessel density compared with the DC-CT26group and the induced HUVEC group(P<0.001,P<0.01).The hemoglobin assay showed that the hemoglobin content of the DC-CT26 group,the induced HUVEC group and the combined group were significantly decreased compared with the PBS group(P<0.001,respectively).The combined group had the lowest hemoglobin content in the tumor tissues compared with the DC-CT26 group and the induced HUVEC group(P<0.001,P<0.01).Western blot showed that the expression levels of tumor vascular endothelial cell specific markers TEM1,TEM8,and VEGFR2 in the induced HUVEC group and the combined group were obviously reduced compared with the PBS group(P<0.001,respectively).The expression levels in the combined group were significantly reduced compared with the DC-CT26 group and the induced HUVEC group,indicating that tumor angiogenesis was minimal in tumor tissues(P<0.001,P<0.01).3.Positive bands were detected in the mice serum of each group at the levels of165 kD,130 kD,and 63 kD by western blot,similarly with the bands produced by the incubation of TEM1,VEGFR2,and TEM8 antibodies with the total protein of induced HUVEC.It was presumed that the positive bands might be TEM1,VEGFR2,and TEM8.4.ELISA assay showed that the HUVEC antibody produced by mice in the induced HUVEC group and the combined group were significantly enhanced compared with the PBS group(P<0.01,respectively).The HUVEC antibody produced by mice in the combined group had the highest titer compared with the DC-CT26 group and the induced HUVEC group(P<0.01,P<0.05).5.The results of mice spleen weight showed that the spleen weight of the mice in the DC-CT26 group,the induced HUVEC group and the combined group were significantly increased compared with the PBS group(P<0.001,P<0.001,P<0.001).The spleen weight of the mice in the combined group was the lightest compared with the DC-CT26 group and the induced HUVEC group(P<0.05,respectively).The spleen index results showed that the spleen index in the DC-CT26group,the induced HUVEC group and the combined group were higher than that of the PBS group(P<0.01,P<0.05,P<0.001).The spleen index in the combined group was the highest compared with the DC-CT26 group and the induced HUVEC group(P<0.01,respectively).6.The results of the killing experiment showed that the CTL killing effect of the DC-CT26 group,the induced HUVEC group and the combined group on CT26 cells were stronger than the PBS group(P<0.001,P<0.01,P<0.001).The CTL of the combined group had the highest killing effect on CT26 cells compared with the DC-CT26 group and the induced HUVEC group(P<0.001,respectively).7.ELISA assay showed that the content of IFN-?in the DC-CT26 group,the induced HUVEC group and the combined group were added compared with the PBS group(P<0.001,respectively).The content of IFN-?in the combined group was the most compared with the DC-CT26 group and the induced HUVEC group(P<0.001,respectively).8.The results of flow cytometry showed that the content of CD3~+CD8~+T lymphocytes in the spleen of the mice in the DC-CT26 group and the combined group were obviously enhanced compared with the PBS group(P<0.01,P<0.001).The content of CD3~+CD8~+T lymphocytes in the spleen of the mice in the combined group was the highest compared with the DC-CT26 group and the induced HUVEC group(P<0.001,respectively).Conclusions1.Induced HUVEC vaccine is superior to HUVEC vaccine in inhibiting mouse colorectal carcinoma.2.The combination of induced HUVEC vaccine and DC-CT26 vaccine has better anti-tumor effect than applying them alone.