Low-dose Paclitaxel Extended-release Phospholipid Gel Combined With Human Papillomavirus Peptide Vaccine For The Treatment Of Cervical Cancer

Objective:Cervical cancer is the fourth most common malignancy in women worldwide.Continuing to be infected with human papillomavirus（HPV）is a key hazard element for the onset of cervical cancer.The currently available HPV vaccine can prevent HPV infection,but it cannot treat HPV infection that has already occurred and cervical cancer induced by HPV infection.Therapeutic HPV vaccines are designed to clear HPV-infected cells and virally transformed tumor cells by inducing an antigen-specific immune response in the body.Our Subject Group has demonstrated that the therapeutic HPV16 E7 peptide vaccine with Cp G ODN as adjuvant can effectively inhibit or remove small volume tumors in early stages.We also found that the vaccine had limited inhibitory effect on medium to late stage large volume tumors。The possible reason for this is that various immune-related interactions in the body become more complex and the role of immunosuppressive factors becomes more powerful the larger the tumor size.In this case,the combination with other anti-cancer modalities including chemotherapy and radiotherapy is a strategy to improve the efficiency of cancer treatment.Paclitaxel,as one of the commonly used chemotherapeutic agents for cervical cancer,has many positive immunomodulatory effects in addition to inducing apoptosis of cancer cells.Low-dose paclitaxel administration has positive immunomodulatory effects such as promoting dendritic cell maturation and suppressing myeloid-derived suppressor cell function;in addition,low-dose rhythmic paclitaxel administration has the advantage of low side effects and low toxicity,while low-dose rhythmic paclitaxel administration demonstrates better therapeutic effects in certain contexts.Therefore,in this study,the combination of low-dose paclitaxel extended-release phospholipid gel and HPV16 E7peptide vaccine with Cp G ODN as adjuvant was used in a C57BL/6 mouse cervical cancer subcutaneous transplantation tumor model to investigate whether the combination therapy could have better therapeutic effect on the Large volume cervical cancer subcutaneous graft tumor,in order to gain more treatment time and opportunity for patients with intermediate and advanced cervical cancer within their precious treatment window.Method:1.Synthetic peptides.The sequence of HPV16 E7 43-77 peptide fragment was GQAEPDRAHYNIVTFCCKCDSTLRLCVQSTHVDIR with>95%purity.2.Constru-ction and treatment of large volume tumor models in mice.C57BL/6 mice were inoculated subcutaneously with TC-1 cells（5×10⁵cells/each）on the back on day 0.At day 13 when the level of tumor size was 150±50mm³,the rats were randomly classified into 4 teams of 6 rats each.The four groups were PBS control group（group I）,paclitaxel extended-release phospholipid gel group（group II）,HPV16 E7 peptide vaccine with Cp G ODN as adjuvant group（group III）,and paclitaxel extended-release phospholipid gel combined with HPV16 E7 peptide vaccine treated with Cp G ODN as adjuvant group（group IV）.On the day after grouping,5 ul of paclitaxel extended-release phospholipid gel was administered intratumorally to mice in the second and fourth groups,and the concentration of paclitaxel in paclitaxel extended-release phospholipid gel was 28 mg/ml.Intratumoral injection of 5 ul of paclitaxel-free phospholipid gel in the first and third groups.A total of 100μl of PBS mixture containing 50μg of E7 peptide with 20μg of Cp G ODN was injected subcutaneously into the right dorsal surface of each rat in the third and fourth groups on day 18.Equal amounts of PBS were administered to the first and second groups.3.To assess the therapeutic effect of combination therapy on tumors.The general living condition of the mice was observed every 2-3 days during the experiment,measurement and documentation of each mice’s tumor size and body weight,the tumor volume growth curve was plotted at the end of the experiment,and the tumor weight was weighed and recorded.4.Detection of the effect of combination therapy on splenic immune cells.Stripping mouse spleen and prepping splenocyte suspension.Detection of white fraction ratios of Tregs（CD4+CD25+Foxp3+）,TAMs（CD11b+F4/80+）,M2-type TAMs（CD11b+F4/80+CD206+）,MDSCs（CD11b+Gr-1+）,M-type MDSCs(CD11b+Ly6G-Ly6C^high)and G-type MDSCs(CD11b+Ly6G+Ly6C^low)cells in spleen tissues of tumor-bearing mice by flow cytometry.5.To detect the effect of combination therapy on TAMs cells in tumor tissues.Tumor tissue is peeled off and suspended solution of tumor cells is made.The percentage of TAMs（CD11b+F4/80+）and M2-type TAMs（CD11b+F4/80+CD206+）cells in tumor tissues of tumor-bearing mice was detected by flow cytometry.6.To detect the effect of combination therapy on local cytokines,chemokines,etc.in tumor tissues.The m RNA levels of TGF-β,IFN-γ,CCL2,CCL5,CCL12,CXCL-9,CXCL-10 and MMP9 were detected in tumor tissues using RT-q PCR technique.8.Statistical analysis.All the data from the laboratory were statistically evaluated in Graph Pad Prism.Evaluating differences in statistics across cohorts used a one-way ANOVA.Tukey’s multivariable check was used for multiple pairwise interviews between clusters.Data are presented as mean±standard deviation.As a guideline for statistical significance,P<0.05.Results:1.Combination treatment effectively inhibits the growth of large-volume cervical cancer in mice.The tumor volume and weight of mice in the combination treatment group were significantly reduced compared with those in the PBS group,The tumor arrest rate was dramatically faster in the combination therapy group than in the PBS band.（P<0.05）.The combination treatment had a significant synergistic effect in inhibiting tumor growth in tumor-bearing mice.2.Combination treatment resulted in a considerable drop in the number of Tregs and M2 TAMs cells in the splenium of mice versus the PBS group（P<0.05）.There was no significant difference in the percentage of total splenic TAMs cells in the combination treatment group compared with the PBS group.3.The number of splenic M-type MDSCs cells was significantly lower in the combination treatment group than in the PBS group（P<0.05）.The ratio of splenic G-type MDSCs cells in the combination treatment group was larger than that in the PBS group（P<0.05）.The difference in the percentage of total splenic MDSCs cells in the combination treatment group compared to the PBS control group was not statistically significant.4.A significant reduction in the proportion of total TAMs cells in tumors of mice in the combination treatment group is found relative to the PBS group（P<0.05）.The percentage of M2 TAMs cells in tumors of mice in the combination treatment group ranged inferior to the PBS group,Values did not meet statistical significance.5.combination therapy can alter the local m RNA levels of multiple cytokines in tumor,The analysis showed that the distribution levels of local IFN-γ,CCL2 and CXCL-9 were significantly higher（P<0.05）and the distribution levels of TGF-β,MMP9,CCL5,CCL12 and CXCL-10were significantly lower（P<0.05）in the tumor of the combination treatment group compared with the PBS group.Conclusion:1.The combination treatment effectively inhibited the growth of intermediate to advanced cervical cancer in mice.2.Combination treatment reduced the number of immunosuppressive cells including Tregs cells,M2-type TAMs cells and M-type MDSCs cells in mouse spleen,and also reduced the number of TAMs cells in mouse tumor,and generally improved the immunosuppressive environment in mouse organism.3.Combination therapy effectively improves the local immunosuppressive microenvironment of the tumor by altering the local expression levels of multiple cytokines and MMP9.