Research On The Preparation Of A Therapeutic Human Umbilical Vein Endothelial Cell Vaccine With Docetaxel As Adjuvant And Its Anti-tumor Effects

Objective:Breast cancer is one of the most common malignant diseases among women,and the incidence rate is increasing year by year,which has caused a serious impact on women's health.During the development and progression of tumors,a large number of new blood vessels will be generated,which will inhibit the growth of tumor by blocking angiogenesis.At present,the commonly used anti-tumor angiogenesis drugs are mostly small-molecule inhibitors and monoclonal antibodies.However,due to their large side effects,short half-life and resistance to drug resistance,their further clinical applications have been limited.Anti-tumor angiogenic vaccine is a new field of anti-angiogenesis therapy.Anti-tumor vaccine treatment has the advantages of wide anti-tumor spectrum,low toxic and side effects,easy to reach the target site of the drug,and low resistance to drug resistance.Previous studies have showed that human umbilical vein endothelial cell(HUVEC)vaccines had antiangiogenic effects.Similar as other tumor vaccines,the antitumor effects of HUVEC vaccine in therapeutic procedure were not satisfactory enough.Therefore,it was necessary to find suitable adjuvant to enhance the anti-tumor effects of HUVEC vaccine.Docetaxel is the first-line chemotherapy drug for treatment of breast cancer and has been widely used in clinical.Studies have verified that low doses of docetaxel could enhance the immune responses of cancer vaccines.This is the first subject which investigate the anti-tumor effects of HUVEC vaccine with low-dose docetaxel as adjuvant.Also,the anti-tumor mechanisms were investigated.Methods:BALB/c mice were randomly divided into the 6 following groups:PBS group,HUVEC group,DOC group(10mg/kg),HUVEC-DOC1(HUVEC vaccine+2.5mg/kg),HUVEC-DOC2(HUVEC vaccine +5 mg/kg),and HUVEC-DOC3(HUVEC vaccine+ 10mg/kg).The subcutaneous xenograft model was established by subcutaneous injection of EMT-6 and 4T1 mouse breast cancer cells to examine the anti-tumor growth effects of the different treatments.An artificial lung metastasis model was established by injecting EMT-6 and 4T1 mouse breast cancer cells into the tail vein.INF-y content in the serum of immunized mice was detected by the ELISA method.The spleen lymphocyte proliferation assay and cytotoxic T lymphocyte killing assay were used to measure the cellular immune response levels of mice treated with different treatments.The mouse spleen was harvested and flow cytometry was used to detect the content of CD8+ T lymphocytes and Treg cells expressing CD4+ Foxp3+ in splenic lymphocytes.Immunofluorescence technique was used to detect the infiltration of CD8+ T lymphocytes in tumor tissue of each group.Immunohistochemical techniques were used to specifically evaluate the formation of tumor microvessels in each group of mice.HE staining was used to detect the pathological changes of tumor tissues in each group.The body weight,hair and daily behavioral activities were used to investigate the safety of different treatment protocols.Results:From the tumor growth curve and weight of tumors in mice,it can be seen that compared with the PBS group,the other groups all inhibited tumor growth to(in)varying degrees,and the HUVEC-DOC2 group had the most significant inhibitory effect.At the same time,compared with the PBS group,the survival time of the mice in the other treatment groups had different degrees of extension,especially in the HUVEC-DOC2 group,the mice had the longest survival time.From the lung metastasis model,we can see that the number of metastases in the HUVEC-DOC2 group was significantly reduced compared with other groups,indicating that this group has the best antitumor effect.The results of T lymphocyte proliferation test showed that the proliferation ability of T lymphocytes in the other four groups,except PBS group and DOC single drug group,was enhanced in varying degrees,especially in HUVEC-DOC2 group,which was the strongest and close to the positive control group.From the results of CTL killing experiments,we found that when the target cells were HUVEC cells,the HUVEC single drug group,HUVEC-DOC1 group,HUVEC-DOC2 group and HUVEC-DOC3 group,the killing ability gradually enhanced with the increase of the effect-target ratio.The HUVEC-DOC2 group had the strongest killing ability.The results of ELISA showed that the levels of INF-y in serum of HUVEC-DOC2-treated mice were significantly higher than those in other groups(P<0.05).From the results of immunofluorescence detection of CD8+ T lymphocyte infiltration in mouse tumors,we found that CD8+ T lymphocyte infiltration in HUVEC-DOC2 treatment group was significantly higher than in other treatment groups.From the results of flow cytometry experiments,the content of CD8+ T lymphocytes in the HUVEC-DOC2 treatment group was significantly higher than that in the other five groups,and the content of Treg cells was most significantly reduced.Compared with other groups,the results of immunohistochemical staining showed that the positive expression level of CD31 in HUVEC-DOC2 group was the lowest,and the microvessels generated in this group were the least.From the results of the safety inspection,it can be concluded that there was no significant difference in body weight,daily behaviors,and diet among the mice in each group.The results of organ biopsy showed that there were no obvious pathological changes in the organs of each group of miceConclusion:Low-dose docetaxel could act as an effective adjuvant to help HUVEC vaccine to display synergistic anti-tumor effects,which could significantly inhibit the continued growth of tumors and tumor angiogenesis.This enhanced anti-tumor effects were accompanied with an enhanced killing capacity of T lymphocytes.The infiltration of lymphocytes in tumors was also significantly enhanced,which might be one reason for the synergistic anti-tumor effects.