| Backgrounds and purposesBackgroundsImmune surveillance is an important function of human immune system that can identify and clear the mutant cells with newborn antigens and virus-infected cells in vivo. During the development of tumors, TSA (tumor specific antigens) and TAA(tumor-associated antigens) are highly expressed on the surfaces of tumor cells, and many TSA or TAA have been screened. However, the immune system fails to kill tumor cells expressing TSA or TAA. In contrast, tumor cells are still in development under the immune system indicating that patients suffered from cancers exist a wide range of immune escape in vivo. Tumor cells avoid recognition and attack by immune system through themselves or tumor microenvironment. The mechanisms of immune escape in tumor are not yet fully understood. The concept of the tumor microenvironment,a more reasonable explanation about mechanisms of tumor escape,has been found. Tumor microenvironment consists of the internal environment in tumor occurrence and development, including tumor cells, interstitial cells, capillaries,tissue fluid and a small amount of infiltrating cells such as DC, macrophages. The immune suppression in tumor microenvironment has many factors: the decreasing number of CD4 + and CD8 + cells in the blood; regulatory T cells increased in the local tumor and adjacent mucosa; DC dysfunction; increasing immunosuppressive factors such as TGF-?; lacking of organization phase compatibility complex of MHC-I, MHC-? molecules. In tumor microenvironment, the above factors are impacted each other. Tumor cells not only evade the attack by immune system passively, but also active inhibition of the normal function of immune cells in the environment.There are two major objectives in cancer treatment: 1) the destruction of visible lesions; and 2) the induction of an immune response capable of destroying micrometastases that are invisible. In the absence of a protective immune response,micrometastases continue to develop into lethal lesions. Because the majority of solid tumors are thought to express TAA, it is believed that the induction of an effective anti-TAA immune response may enable the eradication of micrometastases. The anti-tumor immune include cellular and humoral immumity, and cellular immunity is the main anti-tumor immunity. In recent years, with increasing research of mechanisms of tumor microenvironment, the features and pathological processes of the tumor microenvironment have got a better understanding that DC (dendritic cels)play a key role in initiating the immune response or in induction of immune tolerance in the tumor microenvironment. Tumor vaccines modified by tumor-bearing host DC through various means induce specific killer T cells to generate and stimulate anti-tumor immune function. However, the prognosis of cancers has not been a significant improvement with the application of DC vaccines, even with the application of DC vaccines combined with chemical drugs or other biological agents.Immune therapy can not effectively improve the status of the suppression of immune function in the tumor microenvironment.Blood group antigens may be potential targets to break the tumor microenvironment. ABO blood group, consisting of four types (A, B, O and AB), is one of the most important blood groups for human. The system is mainly determined by the epitopes of A and B antigens on the surfaces of the red blood cells and by anti-A and anti-B antibodies in the serum. The antigens are present not only on red blood cell membranes but also in tissues and body fluids. People of blood type A possess antigen A on the surfaces of cells and anti-B antibody in the serum. Similarly,People of blood type B have antigen B and anti-A antibody. People of blood type AB contain both A and B antigens but no antibodie. While People of blood type O have both antibodies but no antigen. Anti-A and Anti-B antibodies are mostly IgM type.Anti-B, interacting specially with B epitopes on glycolipids and glycoproteins, are absent in people of B or AB but abundant in people of A and O. The distribution of anti-A/B and B/A epitopes in human has been considered to be a major barrier in allogeneil graft transplantation.Review of mechanisms in the ABO blood group antigen-induced hyperacute immune rejection assumes that antigen-induced hyperacute immune rejection can be provoked if the tumor cells express the abnormal blood group antigens. First, unlike the tumor vaccine using tumor antigen to produce antibodies, there are natural antibodies against this antigen, so the immune response may be more rapidly; second,similar to the abnormal blood group antigens, the presenting tumor antigen by APC can be improved in the intratumoral injection of xenoantigen; third, there is reversal of Treg immunosuppressive role in the intratumoral injection xenoantigen. At present,the usage of abnormal blood group antigens to break the tumor microenvironment has not been reported.PurposesAdenovirus vectors expressing blood group B mimic peptides are successfully constructed. The environment of human natural blood group antibodies in mice had been simulated. The injection of these vectors to transplanted tumor subcutaneously in mice can followed by that blood group B antigen mimetic peptides expressed on the surface of tumor cells combine with anti-blood group B antigen-antibodies efficiently and activate the complement-mediated tumor cell killing effect.Intratumoral injection of these adenovirus vectors to the tumor can induce internal inflammation, necrosis, apoptosis of tumor cells and increasing immune cells in tumor.1. Construction, identification and amplification of recombinant adenovirus vectors expressing human blood group B mimic peptidesObjective: To construct adenovirus vectors expressing human blood group B mimic peptides, and to verify its expression in tumor cells.Methods: pShuttle-CMV-DNA was built by a pAdeasy-1 system, and then dephosphorylased by Pme? and CIP. The recombinant plasmid was constructed in E.coli BJ5183 with the adenoviral backbone plasmid by homologous recombination after restriction enzyme digestion by Pac I. The linearized recombinant plasmid was transfected to HEK 293A cells by Lipofectamine 2000 to obtain recombinant adenovirus. Virus titer was determined by cytopathic effect. Expression of mRNA and proteins was analyzed by RT-PCR and Western blot after the infection of B16 cells by these vectors.Results: The adenovirus vectors were analyzed by PCR, restriction enzyme digestion and sequencing analysis. Ad-B/Fas, Ad-B/Fas-IRES-MIP3? were seen about 600 bp inserted fragment, and Ad-MIP3?, Ad-B/Fas-IRES-MIP3? were seen about 297bp specific fragment, but Ad-control has produced no amplified fragments.The sequencing results were as same as the target gene sequence. Recombinant virus titers: Ad-B/Fas: 7 ×1010pfu/ml; Ad-control: 2.21 ×1011pfu/ml; Ad-MIP3?:1.2 ×1011 pfu/ml; Ad-B/Fas-MIP3?: 3.94 ×1010pfu/ml. The mRNA and protein were expressed after infection of B16 cells.Conclusion: We successfully constructed the recombinant adenovirus vectors edcoding the gene of the human blood group B antigen mimetic peptides. The mRNA,protein can be efficiently expressed in B16 cells after infection by adenovirus vectors.2. Anti-tumor effect of recombinant adenovirus vectors encoding human blood group B antigen mimic peptides gene in vitroObjective: Anti-tumor effect of recombinant adenovirus vectors expressing human blood group B mimic peptipes in vitro.METHODS: The experiment is divided into five groups: Ad-B/Fas group,Ad-MIP3? group, Ad-B/Fas-MIP3? group, Ad-control group and PBS group. B16 cells were infected with adenovirus vectors, Apoptosis was tested by flow cytometry in vitro (6 cases per each group, a total of 30 cases). Cell proliferation assay was tested by MTT (Each group is divided into three subgroups, each subgroup consists of 12 cases, a total of 180 cases). Complement-mediated cell killing and cell antibody-dependent cell-mediated cytotoxicity experiments were tested by MTT(Each group is divided into three subgroups, each subgroup consists of 8 cases, a total of 120 cases). Apoptosis data was analyzed by one-way ANOVA analysis.Bonferroni group comparisons was tried if heterogeneity of variance, or selecting the approximate F-test Welch method and Dunnett'S T3 groups if not. The remaining measurement data using the factorial design variance analysis, P<0.05 statistically significant. All statistical analyzes using SPSS 13.0 software package.Results: (1) Apoptosis rate in each treatment group was statistically significant(F=13.586, P<0.001). Compared with apoptosis rate of PBS group (2.62± 0.70)%,apoptosis rate of Ad-B/Fas (5.68 ± 1.31)% and Ad-B/Fas-MIP? (5.94 ± 1.21)% had statistically significant difference (P<0.001); While apoptosis rate of Ad-control group (2.62±1.19)% and Ad MIP3p (3.55±0.86)% had no significant difference(P=1.000); apoptosis rate between Ad-B/Fas and Ad-B/Fas-MIP3? had no significant difference (P=1.000); Apoptosis rate of Ad-B/Fas group was higher than that of Ad-control group or Ad the-MIP3? group or PBS group (P<0.001; P=0.022; P<0.001).(2) MTT showed that significant differences between the different adenovirus vectors were significant (F=1165.189, P<0.001). Compared with cell proliferation of PBS group, cell proliferation of other groups was statistically significant (P< 0.001).Compared with cell proliferation of Ad-control group, cell proliferation of other three virus groups was not statistically significant (P=0.986; P=1.000; P=0.876). Different concentrations of groups had a significant difference (F=6292,979,P<0.001). (3)CDC experiments showed significant differences among different adenovirus vectors(F=107.762, P<0.001). Compared with the PBS group, CDC effect in group of Ad-B/Fas, group of Ad-control, group of Ad-MIP3P, group of Ad-B/Fas-MIP3? was statistically significant (P<0.001). Compared with Ad-control group, CDC effect in group of Ad-B/Fas, group of Ad-MIP3? and group of Ad-B/Fas-MIP3? had a significant difference (P<0.001); CDC effect in group of Ad-B/Fas was greater than the other four groups (P<0.001). There were significant difference among the different concentrations of complement (F=52.998, P<0.001); CDC effect in group of complement 5ul had a significant difference with the other two groups (P<0.001),There was no significant difference in CDC effect between complement lOul and 20ul (P=1.000). (4) There were significant differences in ADCC effect among the different adenovirus vectors (F=108.050, P<0.001). Compared with the group of PBS, ADCC effect in group of Ad-B/Fas, group of Ad-control, group of Ad the-MIP3? and group of Ad-B/Fas-MIP3? had significant difference (P<0.001);Compared with group of Ad-control, ADCC effect in group of Ad-B/Fas, group of Ad-MIP3? and group of Ad-B/Fas-MIP3? had a significant difference (P<0.001);ADCC effect in group of Ad-B/Fas was significant different with the other four groups(P<0.05). There were significant differences between different effector target ratio (F =54.542, P<0.001); Compared to the other two groups, ADCC effect in group of 10:1 was lower (P <0.001), There was no significant difference in ADCC effect between 20:1 and 40:1 (P= 1.000). The interaction effect between group and T ratio was statistically significant (F=2.882, P=0.006). (5) Antigen uptake and phagocytic effects of tumor cell antigens by DCs and macrophage in groups of Ad-B/Fas and Ad-B/Fas-MIP3? were greater than those in goups of Ad-control group and Ad-MIP3p.Conclusion: B16 melanoma cells were infected with adenovirus encoding blood group B peptide genes. Mechanisms in destruction of tumor cells may be explained by apoptosis, CDC, ADCC and increasing uptake and phagocytosis of tumor cell antigens by DC and macrophages.3. Anti-tumor effect of recombinant adenovirus vectors expressing human blood group B mimic peptides in vivoObjective: To construct a subcutaneously transplanted melanoma model after simulating the human blood group B antibodies environment in C57/BL6 mice. To observe the tumor growth in mice and impact on tumor microenvironment by intratumoral injection of recombinant adenoviral vectors expressing human blood group B mimic peptides.Methods: the mice was randomly divided into five groups: Ad-B/Fas group;Ad-control group; Ad-mip3? group; Ad-B/Fas-mip3? group; PBS group (N = 8, a total of 40). Tumor volume and survival time of mice were observed respectively;Apoptosis rate of transplanted tumor cells was tested by flow cytometry;Tumor-infiltrating lymphocytes of CD3+,CD4+,CD8 + and CD4 + CD25 +subsets were detected by flow cytometry; IL-2, IFN-gamma and TNF-a were detected by ELISA assay; The expression of CD34, VEGF, CD49b, CD68, CD80 and CD86 was detected by immunohistochemistry of transplanted tumors, and expression of MHCI,CD11c was detected by immunofluorescence; The mice were divided to five groups using the cells infected with the viruses before transplantation (N= 8, a total of 40) ; Tumor attacking after immunization with killed B16 cells infected with the viruses(N=8, a total of 40); experiments combined with microwave ablation(Combined with MA experiment were randomly divided into six groups:MA+Ad-B/Fas intratumoral injection group, MA+Ad-control intratumoral injection group and MA group, Ad-B/Fas intratumoral injection group, the Ad-the control PBS group intratumoral injection group,n=8, a total of 48). Tumor volumes of different groups at different times were repeated measurement with applications SPSS13.0 statistical package. Other data were used one-way ANOVA analysis. Bonferroni were used if heterogeneity of variance. F-test Welch and Dunnett's T3 were used if not heterogeneity of variance. Each group of mice survival was used for theKaplan-Meier method.Results: (1) the difference of effect between the groups was statistically significant (F=14.701, P<0.001). Compared with the group of PBS, tumor volume in group of Ad-control and group of Ad-MIP3? was not significant different (P=0.370;P=0.092), tumor volume in group of Ad-B/Fas and group of Ad-B/Fas-MIP3p was significant different (P=0.007; P=0.007). Tumor volume in group of Ad-B/Fas and Ad-B/Fas-MIP3? was slower than other groups. (2) Mice in each group were set to observation the time limited within 60 days. Median survival in Ad-B/Fas group was 30 (95% CI :25.842-34.158) days, Ad-MIP3p group of 23 (95% CI :17.456-28 .544)days, Ad-B/Fas-MIP3p group of 33 (95% CI: 30.228-35.772) days, Ad-control group of 21 (16.842-25.158) days, PBS group of 21 (95% CI :18.228-23 .772) days.Compared with group of PBS, survival time of mice in group of Ad-B/Fas and group of Ad-B/Fas-MIP3? was significant different (P=0.006; P<0.001), and survival time of mice in group of Ad-control and group of Ad-MIP3? was not significant different(P=0.368; P=0.134). Compared with group of Ad-control, survival time of mice in group of Ad-B/Fas-MIP3? was significant different (P=0.002), but survival time of mice in group of Ad-B/Fas was not significant different (P=0.052); There was no significant difference between the group of Ad-B/Fas and group of Ad-B/Fas-MIP3?(P=0.989). (3) Intratumoral injection Ad-B/Fas resulted in tumor cell necrosis,lymphocytes and other inflammatory cell infiltration. And intratumoral injection of Ad-B/Fas-MIP3? resulted in tumor tissue necrosis and inflammatory cell infiltration.The remaining three groups of mice tumor tissue can be seen a small amount of tumor necrosis and inflammatory cell infiltration. There was no significant inflammatory response in the two groups of lungs, liver, intestines, heart, kidney, spleen and other organs (4) Effect of tumor cell apoptosis in different groups was statistically significant (F=77.696, P<0.001). compared with in group of PBS,apoptosis in group of Ad-control was not significant different (P=0.571), tumor cell apoptosis in group of Ad-B/Fas, group of Ad-MIP3?, group of Ad-B/Fas- MIP3? had significant difference (P<0.001). Compared to group of Ad-B/Fas, apoptosis in group of Ad-B/Fas- MIP3? was no significant different (P=0.547), apoptosis in group of Ad-control and group of Ad the-MIP3? was statistically significant (P<0.001;P<0.001);There were a lot of necrosis in group of Ad-B/Fas. (5)Lymphocyte-CD3,CD8 subsets was no significant different by flow cytometry (F=1.684, P=0.185;F=0.699, P=0.600). The experimental group of lymphocytes, CD4, CD25 and CD4/CD8 subsets was statistically significant (F=7.399, P<0.001; F=18.542, P<0.001 and F=5.327, P=0.003). Compared with PBS group, CD4+ cell in Ad-B/Fas group increased statistically significant (P=0.003), and there was no significant difference in the remaining three groups of CD4 + lymphocyte (P=1.000). There was no significant difference group of Ad-B/Fas and group of Ad-B/Fas-MIP3? in CD4 ?lymphocyte count (P=0.154). Compared with the group of PBS, CD25+ cell count in group of Ad-B/Fas, group of Ad-B/Fas-MIP3? was statistically significant declined(P<0.001),CD25+ cell count in group of Ad-control, group of Ad the-MIP3? was statistically significant (P=1.000; P=0.217). There was no significant difference in CD25 + cell number in group of Ad-B/Fas and group of Ad-B/Fas-MIP3? (P=0.412).Compared with the group of PBS, CD4/CD8 in group of Ad-B/Fas increased statistically significant (P=0.012), and the difference of CD4/CD8 in the remaining three groups was not statistically significant (P=1.000). (6) There was significant different in IL-2 among experimental groups (F=119.620; P<0.001). Compared with the PBS group, IL-2 in group of intratumoral injection of adenoviral vectors improved statistically significant (P<0.001; P=0.003; P=0.003; P<0.001); compared with Ad-control group, Ad-B/Fas and Ad-B/Fas-MIP3? group increased statistically significant in IL-2 (P<0.001). Compared with the PBS group, group in Ad-B/Fas,Ad-control, Ad-B/Fas-MIP3p improve the IFN-y in a statistically significance (P<0.001; P=0.006; P<0.001); compared with Ad-control group, IFN-y in Ad-B/Fas group improved in a statistically significant (P< 0.001); compared with the PBSgroup, TNF- a in each adenovirus group improved in statistical significance (P<0.001); compared with Ad-control group, TNF-a in Ad-B/Fas, Ad-MIP3? and Ad-B/Fas-MIP3p was statistically significant (P=0.001; P=0.013; P=0.003). (7) There was no significant of differences in CD86 optical density among groups (F=2.287,P=0.088). The effect in the rest of variable group differences were statistically significant (P<0.05). Compared with the group of PBS, difference of CD34 opticaldensity in group of Ad-B/Fas, group of Ad the-MIP3? group, group of Ad-B/Fas-MIP3p was statistically significant (P<0.001; P= 0.002; P<0.001), and the difference of CD34 optical density in group of Ad-control was no significant(P=0.800). The difference of CD34 optical density in group of Ad-B/Fas group and Ad-B/Fas-MIP3? was no significant (P= 1.000). Compared to the PBS group, the difference of VEGF optical density in group of Ad-B/Fas, group of Ad-MIP3p, group of Ad-B/Fas-MIP3p was statistically significant (P<0.001; P=0.020; P<0.001), and the difference of VEGF optical density in group of Ad-control was not statistically significant (P=0.209). The difference of VEGF optical density between group of Ad-B/Fas and group of Ad-B/Fas-MIP3p was significant different (P=0.001), and the difference of VEGF optical density between group of Ad-B/Fas and group of Ad-MIP3? was not significant different (P=0.516). Compared with the PBS group,the difference of CD68 optical density in the remaining four groups was statistically significant (P<0.001). Compared with group of Ad-control, the difference of CD68 optical density in group of Ad-B/Fas, group of Ad-MIP3?, group of Ad-B/Fas-MIP3?was not statistically significant (P=0.216; P=1.000; P=0.763). Compared with the group of PBS, the difference of CD80 optical density in group of Ad-control, group of Ad-MIP3?, group of Ad-B/Fas-MIP3? group was not statistically significant(P=1.000), the difference of CD80 optical density in group of Ad-B/Fas was statistically significant (P<0.001). Compared with the group of PBS, the difference of CD49b optical density in group of Ad-B/Fas, group of Ad-MIP3?, group of Ad-B/Fas-MIP3? was not statistically significant (P=1.000; P=1.000; P=0.083), and the difference of CD49b optical density in group of the group of Ad-control was significant different (P=0.006). Compared with the PBS and Ad-control group, the CD11c and MHCI immunofluorescence results in group of Ad-B/Fas were higher. (8)Each group of mice was 100% successful inoculated with the corresponding tumor cell suspension into the tumor. The between-group effect of tumor formation time was statistically significant (F=8.782, P<0.001). Compared with Ad-control group,Tumor formation time in Ad-B/Fas group is longer statistically significant (P=0.041).Compared with Ad-control group and group of Ad-B/Fas without immunization with B red blood cells, prolonged survival in Ad-B/Fas group was statistically significant(P<0.001). The effect of the difference in tumor volume between the two groups was statistically significant (F=3.521, P=0.016). there are significant differences in each treatment factors on the size of mice. Differences in repetition effects were statistically significant (F=80.349, P<0.001), the tumor volume of each measurement point in time there are differences with the increase of the point in time. the mice tumor volume gradually increased. The packet processing factors on the tumor volume increased significant difference in between interaction effect (F=3.694,P=0.012). (9) the constituent tumor formation time of the group effect was statistically significant (F=8.972, P<0.001). Compared with the group of Ad-B/Fas without immunization with B red blood cells and Ad-control group, tumor formation time and lifetime in Ad-B/Fas was statistically significant (P=0.011; P=0.008).Compared with group of Ad-B/Fas, tumor formation time in group of Ad-MIP3? and group of Ad-B/Fas-MIP3? was not significant different (P =0.068; P=0.844).Compared with the group of Ad-control, survival in group of Ad-B/Fas was significant different (P=0.007).The difference of effect between the tumor group was statistically significant (F=7.732, P< 0.001). There are significant differences in each treatment factors on the size of mice. Differences in repetition effects were statistically significant (F=31.682, P < 0.001). (10) Intratumoral injection of adenoviral vectors combined with microwave ablation experiments show that: each group of mice were dead in 60 days. there is a significant difference in survival time between PBS group and Ad-B/Fas(P=0.006). There was not statistically significant between the groups combined with microwave ablation and groups not combinedwith microwave ablation. There was statistically significant in the effect of the difference in tumor volume between the two groups (F=29.247, P<0.001). There are significant differences in each treatment factors on the size of mice. Differences in repetition effects were statistically significant (F=127.957, P<0.001). There are differences in the tumor volume of each measurement point. With the increase of the point in time, the mice tumor volume gradually increased. There was nosignificant difference among the three groups at all time points combined with microwave ablation group (F=52.465, P<0.001).Conclusion: The successful simulation of blood group antibodies in C57/BL6 mice was built before melanoma xenograft model. With intratumoral injection of adenovirus vectors expressing blood type B mimic peptides, The tumor burden was reduced and survival of mice was prolonged. Its possible mechanism may be the intratumoral injection of adenovirus vectors inducing tumor cell apoptosis,enhancement on specific and nonspecific cellular immunity, increasing the secretion of inflammatory cytokines, reduction in tumor microvascular, enhancement on APC antigen-presenting function. Combined with microwave ablation could significantly reduced the tumor burden. |
PDF Full Text Request