| Objective: Cervical cancer is one of the most common cancers among females.The metastasis of cervical cancer has been identified as an important prognostic factor affecting its prognosis.The routes of metastasis include direct spread,lymphatic metastasis and haematogenous metastasis.Data show that 4.16%-7.7% of cervical cancer patients have lung metastases due to haematogenous metastasis.Compared with patients with local cervical cancer,the five-year survival rate of patients with metastatic cervical cancer is significantly reduced,only 16.5%.Surgery is a treatment choice that can provide survival advantages for patients with metastatic cervical cancer,but it will be affected by the number and size of the patients’ pulmonary metastasis,the patients’ own conditions(such as whether there is lymphatic metastasis).Patients with fewer than two pulmonary metastases had a significant survival advantage after pulmonary metastasectomy.Moreover,further chemotherapy is needed after surgery to improve the patient’s prognosis.Although ablation therapy or a combination of radiotherapy and chemotherapy can prolong the patient’s survival to a certain extent,its side effects significantly reduce the patient’s quality of life.Therefore,it is imperative to develop a safe and effective way to treat cervical cancer metastases.Our previous research confirmed that HPV polypeptide vaccine with CpG ODN as an adjuvant can effectively induce cellular immune response in cervical cancer model mice,reduce the number of systemic and tumoral local immunosuppressive cells,reduce tumor local blood vessel formation,and then inhibit the growth of subcutaneous transplanted tumors and cervical tumors in situ.In this study,we established model of pulmonary metastases originating from cervical cancer to evaluate whether subcutaneous vaccination can control the formation and progression of lung metastases,and the immune mechanism of the vaccine.The results of this study will provide a new treatment mode or adjuvant treatment mode for patients with cervical cancer metastases.Method: 1.Synthesis of peptides.The HPV16 E7 43-77 peptide was synthesized by GL Biochem(Shanghai)Ltd.(Shanghai,China).Its purity was > 95%.2.Tumor challenge and immunization.TC-1 cells transformed with HPV16 E6,E7 and Ras genes were implanted into C57BL/6 mice via tail vein injection to establish a model of pulmonary metastases originating from cervical cancer.Female C57BL/6 mice aged 6-8 weeks were randomly divided into four groups,6 mice per group(i.e.,control,Cp G ODN alone,E7 peptide alone,and vaccine group).They were immunized with PBS,Cp G ODN(20μg per mouse),E7 43-77(50μg per mouse),and 20μg of Cp G ODN mixed with 50μg of E7 43-77,respectively.Each mouse received a total of three immunizations at 7day intervals.3.Analysis of anti-pulmonary metastases effect induced by vaccination.At 30 days following tumor cells challenge,the mice were euthanized and lung tissues were obtained.Lung weight was measured and lung nodules were counted to evaluate the anti-tumor effect of the vaccine on mouse cervical cancer lung metastases.4.Analysis immune responses induced by the vaccination in the lungs.At 30 days following tumor cells challenge,the mice were euthanized and lung tissues were obtained.CD4+ T cells and CD8+ T cells in the lung tissues were detected by flow cytometry.5.Evaluating the effect of vaccine on immunosuppressive cells in the lungs.At 30 days following tumor cells challenge,lung tissues from mice were harvested,and a single-cell suspension was prepared to detect the populations of MDSCs(CD11b+Gr-1+),TAMs(CD11b+F4/80+)and Tregs(CD4+Foxp3+)cells by flow cytometry.6.Detecting the effect of the vaccine on the expression levels of cytokines,chemokines and matrix metalloenzymes in the lungs.At 30 days following tumor cells challenge,lung tissues from mice were harvested to detect cytokines(IFN-γ,IL-10)and chemokines(CCL2,3,5,12,19,CXCL-1,CXCL-8)and the expression level of matrix metalloenzymes(MMP2,3,7,9)by real-time fluorescent quantitative PCR.7.Analysis of effect of the vaccination on tumor-infiltrating immune cells,cell proliferation,and p53 expression in the lungs.At 30 days,lung tissues were obtained to detect tumor-infiltrating CD4+ and CD8+T cells and then analyze the expression of Ki67 and p53 by immunohistochemical staining.8.Statistical analysis.All statistical analyses were performed using the Prism software version 7.Differences between all the groups were assessed through one-way analysis of variance(ANOVA),followed by Tukey’s multiple comparison test.Data are represented as the mean ± standard deviation.P-values < 0.05 denoted statistically significant differences.Results:1.Anti-pulmonary metastases effect induced by subcutaneous administration of the vaccine.Compared with the PBS group,the number of lung tumor nodules andthe weight of lung tissue in the vaccine group were significantly reduced.The number of lung tumor nodules in the Cp G ODN group also significantly decreased.There was no significant change in the HPV 16 E7 peptide group.2.Immune responses induced by the vaccination.The percentage of CD4+ T cells and the percentage of CD8+ T cells in the vaccine group were significantly higher than those in the PBS group and the E7 peptide group.It was also found that the percentage of CD4+ and CD8+ T cells in the Cp G ODN group increased,and the percentage of CD8+ T cells in the E7 peptide group increased,but the difference was not statistically significant.3.Inhibition of immunosuppressive cells induced by the vaccination.Compared with the PBS group,the percentage of MDSCs in the Cp G ODN group,the E7 polypeptide group alone and the vaccine group decreased significantly.The percentage of TAMs in the E7 peptide alone and vaccine groups decreased significantly,but the difference between the two groups was not statistically significant.There was no statistically significant difference in the percentage of Tregs between the groups.4.The effect of the vaccine on the expression levels of cytokines,chemokines and matrix metalloenzymes in lung tissue.Compared with the PBS group,the expression level of IFN-γ in the vaccine group significantly increased,and the expression of IL-10,CCL2,3,5,12,19,CXCL-1,CXCL-8 and MMP2,3,7,9 significantly reduced.The expression of each index of Cp G ODN and E7 peptide group was different.5.Effect of the vaccination on tumorinfiltrating immune cells,cell proliferation,and p53 expression.Compared with the PBS group and E7 peptide group,the infiltration of CD4+ T cells and CD8+ T cells in the vaccine group increased significantly.The expression of Ki67 and p53 decreased significantly in the Cp G group and the vaccine group.Conclusion:1.Subcutaneous vaccination can inhibit the growth of cervical cancer lung metastases.2.Vaccination can increase the infiltration of CD4+ T cells and CD8+ T cells in the lungs;reduce the infiltration of MDSCs and TAMs.3.Vaccination can change the expression levels of a variety of cytokines,chemokines and MMPs in the lungs. |
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