| BackroundIt is now little disputed that most if not all cancer cells express antigens that can be recognized by specific T lymphocytes.Lymphocytes of immune system can kill the tumor cells directly within the tumor.Every stages of the process that lymphocytes killing tumor cells can be observed,without accompanying tumor rejection,and clinical responses have been infrequent.The function of effector immune cells is suppressed at the effector phase of the anti-tumor immune response. Tumor can escape from the rejection by immune system,the mechanisms of which are not completely clarified.The concept of tumor microenvironment is proposed by some investigators,and this concept can explain why the tumor cells grow continously regardless of being attacked by immune cells reasonably.Tumor microenvironment is the environment in which tumor cells can make progress, consisting of tumor cells,mesenchymal cells,tissue fluid,microvessel and a few infiltration cells such as dendric cells and macrophages and so on.Tumor microenvironment is the execution place in which anti-tumor immune response occured.Host's immune system has the ability to initiate the anti-tumor immune response,and then generate effector immune cells.These effector immune cells enter into the tumor microenvironment to kill tumor cells.Tumors have the ability to foster a tolerant microenvironment and the activation of a plethora of immunosuppressive mechanisms,which may act in concert to counteract effective immune responses. Immunosuppressive state within the tumor microenvironment is not reversed by regulating total immune system function of patients.Trying to relieve the immunosuppressive factors in tumor microenvironment may elicit an effctive immune-mediated destruction of tumor cells.Chronic inflammatory response can lead to malignant transformation of normal cells through the DNA damage.At present,long-term incidence of inflammation stimulate the tumor has become the conclusion.But the inflammatory response of immune cells gathering also not conducive to tumor development mechanism.It is useful for the treatment of tumor that acute intlammatory can attract immune cells, especially APC and T cells.Antigen substance which belong to different species as compared to host known as xenogeneic antigen.Under normal circumstances, because of the immunogenicity of heterologous antigens are relatively strong,easily lead to a stronger immune response.Natural antibody-mediated hyperacute rejection are the biggest obstacle to clinical xenotransplantation.Xenotransplantation may occur by natural antibody-mediated,complement-dependent cytotoxic effect, resulting in hyperacute rejection happened.Some scholars have already made use of natural antibodies pre-existing in the body to tumor immunotherapy.Human erythrocyte membrane contains a variety of antigens,including many carbohydrate antigen,peptide antigen,and so on.Human erythrocyte membrane are xenoantigens for mice.Existence of natural antibody agaist human erythrocyte membrane antigens in mice is possible.Intratumoral injection of human erythrocyte membrane may lead to a similar effect to hyperacute rejection mediated by natural antibody and complement-dependent rejection effect.Without or even a relatively low level of natural antibodies exist in mice,the heterologous antigen can stimulate the immune system in mice,resulting in induction of corresponding antibodies,resulting in complement-dependent cytotoxicity,manifested as a similar effect of delayed xenograft rejection.Xenogeneic antigens can be recognized by T cells in mice,and consequent immune response can also cause acute or chronic xenograft rejection effect.The above mechanisms may lead to immune response within tumor in mice, produce complement fragments with chemotactic activity,a variety of immune cells chemotaxis into the tumor,and yield a significant inflammatory response within tumor.The number of immune cells,antigen-presenting cells increase,and secrete various cytokines,which activate more immune cells.Non-specific inflammatory response can cause necrosis of tumor cells,in turn trigger the anti-tumor specific immune response,thereout reverse the tumor microenvironment immunosuppressive status and achieve the purpose of control of tumor growth.ObjectiveThe aim of this investigation was to determine the antitumor efficacy of a kind of immunotherapy method of intratumoral injection of human erythrocyte membrane of blood group type A which are xenoantigens for the mice and to observe if this method can obviously lead to the tumor rejection effects and the changes of immune status within the tumor microenvironment.The possible anti-tumor mechanisms of this kind of immunotherapy method were preliminary explored.The ultimate goal was to develop a simple and effective tumor immunotherapy method.Methods1.Human erythrocyte membrane of blood group type A was produed by a hypotonic lysis method.Thirty-five healthy Kunming mice were randomly divided into two major groups,twenty mice were immunized by 0.2ml of 2%suspension of human erythrocyte of blood group type A,intraperitoneal injection once a day,twice a week, for two weeks,fifteen mice were not immunized.Anti-blood group antigen A antibody titer was detected.Kunming mice bearing S180 sarcoma model were established.Five immunized mice were subcutaneously injected a total of 0.1ml human erythrocyte membrane of blood group type A with a concentration of 5mg/ml dissoved in 0.9%Sodium chloride simultaneously when S180 sarcoma model were established.After the sucessful formation of subcutaneous tumors,ten tumor bearing mice seleceted from non-immunized mice were divided into two groups:the intratumoral injection of saline group(the control group) and the intratumoral injection of human erythrocyte membrane of blood group type A group,five mice of each group.Ten tumor bearing mice seleceted from non-immunized mice were divided into two groups:the intratumoral injection of saline group and the intratumoral injection of human erythrocyte membrane of blood group type A group, five mice of each group.The mice were treated with a total of 0.1ml of human erythrocyte membrane of blood group type A suspension with a concentration of 5mg/ml dissolved in 0.9%saline or 0.9%saline according to the different groups, once a day,for five days.The greatest length and width of vertical length were measured by vernier caliper before the first injection and three days,seven days,fourteen days after the first injection.Tumor volume were calculated.2.Seventy healthy Kunming mice were immunized and subcutaneous S180 sarcoma model were established according to the mehtods mentioned above.Sixty mice were randomly divided into four groups:the intratumoral injection of saline group(control group) and the intratumoral injection of human erythrocyte membrane of blood group type A group(non-immunized),fifteen mice of each group,the intratumoral injection of saline group(immunized) and the intratumoral injection of human erythrocyte membrane of blood group type A group(immunized),fifteen mice of each group.Different groups were treated as before.Fourteen days after the first injection, six mice selected from each group were sacrificed by cervical dislocation method, tumors were removed,resected and sectioned.Histology examination of treated S180 sarcoma lesions was conducted by HE staining method to observe tumor necrosis, and infiltration of inflammatory cells.Survival time were observed in the remaining nine mice in each group.3.Tumor infiltrating lymphocytes and tumor cells were seperated by discontinuous density gradient centrifugation method.Apoptosis rates of tumor cells were analyzed by Annexin-V/PI staining method.Expression of MHC-I antigen on tumor cells and proportions of CD3~+CD8~+,CD3~+CD4~+and CD3~+CD4~+CD25~+subsets of tumor infiltrating lymphocytes were detected by flow cytometry menthod.Contents of IL-2, IFN-γ,TNF-α,C5a within the tumor were determined by ELISA assay.Apoptosis indexes of tumor cells were calculated by TUNEL assay.CD34 staining of vascular endothelial cells by immunohistochemical staining,and microvessel densities within the tumor in different groups were caculated.4.Statistical methods:(1)Statistical comparisons of tumor volumes in different groups at different times were made using ANOVA for the repeated measures. Statistical comparisons of tumor volumes at same measure time point in different groups using one-way ANOVA and LSD method was selected for multiple comparisons,if equal variances not assumed,Welch test and Dunnett's T3 multiple comparisons was selected.(2)Statistical comparisons of survival times of mice in different groups were made using Kaplan-Meier test.(3)The others statistical comparisons were made using 2×2 factorial analysis,independent-samples T test was used for simple effects analysis.Results1.Antibody titer in mice serum against blood group antigen A was 1:128.2.Tumor volumes in different groups increased gradually along with the time(P＜0.01).There were significant different influence on tumor volumes increasing in different treatment factors(P＜0.01),and the grouping factor and the repeat factor had an interaction effect(P＜0.01).Tumor volumes in different groups had not significant difference before treatment(P＞0.05).Tumor volumes in different groups demonstrated significant difference 14 days after the first treatment(P＜0.01).Tumor volumes in the human erythrocyte membrane of blood group type A intratumoral injection group,the human erythrocyte membrane of blood group type A inoculated simultaneously with the tumor cells group,the human erythrocyte membrane of blood group type A intratumoral injection group(immunized) were smaller than those in the control group(P＜0.05),but there were no significant difference in tumor volumes between the saline intratumoral injection group(immunized)and the control group.3.The tumor bearing mice immunized by human erythrocyte of blood group antigen A reduced tumor weights(P＜0.05),Intratumoral injection of human erythrocyte membrane of blood group type A also reduced tumor weights(P＜0.01).There was no interaction effect between the immunization factor and the injection factor(P＞0.05). Tumor weights reducing caused by intratumoral injection of human erythrocyte membrane of blood group type A were not influenced by immnization or not.4.Tumor necrosis,infiltration of inflammatory cells such as lymphocytes were observed in tumor tissues section examination after the intratumoral injection of human erythrocyte membrane of blood group type A,especially when the mice immunized by human erythrocyte of blood group antigen A.However,the same circumstance was not observed after the intratumoral injection of saline.5.The mice survival times in different groups showed no statistical difference(P＞0.05).Median suvival time was 24.0(18.156 29.844)days in the control group, 29.0(23.456 35.544)days in the human erythrocyte membrane of blood group type A intratumoral injection group,25.0(18.070 31.930)days in the saline intratumoral injection group(immunized),29.0(23.156 34.844)days in the human erythrocyte membrane of blood group type A intratumoral injection group(immunized).6.Apoptosis rates showed no significant difference whether immunized by human erythrocyte of blood group antigen A or not(P＞0.05).Intratumoral injection of human erythrocyte membrane of blood group type A significant induced tumor cells apoptosis(P＜0.01).Tumor cells apoptosis caused by intratumoral injection of human erythrocyte membrane of blood group type A were not influenced by immnization or not(P＞0.05).7.Expression rates of MHC-â… antigens on tumor cells were not influenced by the immunization factor(P＞0.05).Intratumoral injection of human erythrocyte membrane of blood group type A significant upregulated the expression rates of MHC-â… antigens on tumor cells(P＜0.01),and this kind of upregulation effect was not associated with immnization or not(P＞0.05).8.Quantities of CD3~+CD8~+CTL cells within the tumor were not influenced by the immunization factor or the intratumoral injection factor(P＞0.05).But both the two kind of factors demonstrated significant impact on quantities of CD3~+CD4~+Th cells wihtin the tumor(P≤0.01).There was no interaction effect between the two factors(P＞0.05).Both intratumoral injection of human erythrocyte membrane of blood group type A or intraperitoneal injection of human erythrocyte of blood group antigen A could increase the quantities of CD3~+CD4~+Th cells within the tumor. Quantities of CD3~+CD4~+Th cells within the tumor in the human erythrocyte membrane of blood group type A intratumoral injection group(immunized) were higher than those in the other three groups.The two factors reduced the quantities of CD3~+CD4~+CD25~+Treg cells within the tulmor(P＜0.05),but there was no interaction effect between the two factors(P＞0.05).Quantities of CD3~+CD4~+CD25~+Treg cells within the tumor in the human erythrocyte membrane of blood group type A intratumoral injection group(immunized) were lower than those in the other three groups.9.Both the immunization factor and the intratumoral injection factor could increase the contents of IL-2 within the tumor(P＜0.05),but there was no interaction effect between the two factors(P＞0.05).Highest contents of IL-2 within the tumor in the human erythrocyte membrane of blood group type A intratumoral injection group(immunized) were detected.Both the immunization factor and the intratumoral injection factor showed no influence on contents of IFN-γor TNF-αwithin the tumor(P＞0.05).Neither the immunization nor the intratumoral injection of human erythrocyte membrane of blood group type A 14 days after the first treatment increased the contents of IFN-γor TNF-αwithin the tumor.10.The immunization factor showed no statistical significance on contents of C5a within the tumor(P＞0.05).Intratumoral injection of human erythrocyte membrane of blood group type A significant increased the contents of C5a within the tumor(P＜0.01).There was no interaction effect between the two factors(P＞0.05).11.The immunization factor showed no statistical significance on the apoptosis indexes of tumor cells(P＞0.05).Intratumoral injection of human erythrocyte membrane of blood group type A significant increased the apoptosis indexes of tumor cells(P＜0.01).There was no interaction effect between the two factors(P＞0.05).Compared with the saline intratumoral injection group,more apoptotic cells were observed in the human erythrocyte membrane of blood group type A intratumoral injection group,and these apoptotic cells were distributed surrounding the necrotic cells in most cases.12.The immunization factor showed no statistical significance on the microvessel densities within the tumor(P＞0.05).Intratumoral injection of human erythrocyte membrane of blood group type A significant reduced the microvessel densities within the tumor(P＜0.01).There was no interaction effect between the two factors(P＞0.05).Conclusions1.Intratumoral injection of heterologous erythrocyte membrane had the ability to inhibit tumor growth of S180 sarcoma bearing mice.2.Intratumoral injection of heterologous erythrocyte membrane gave rise to obvious tumor cells necrosis and apoptosis,inflammatory cells infiltration.3.The mechanisms of anti-S180 sarcoma ability for heterologous erythrocyte membrane were related to complement activation,increasing of IL-2 content and the quantities of CD3~+CD4~+Th cells,upregulation of MHC-â… antigens on tumor cells, reducing of tumor microvessel indensity and the quantities of CD3~+CD4~+CD25~+ Treg cells within the tumor microenvironment. |
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