Combination Of CTLA-4and JAK2Blockade To Treat Breast Cancer In Mice Model

Background and objective:Breast cancer is the most frequent cancer in women worldwide.The traditionalmethod of treatment make not a huge trauma to the patient, and the nidus ofmicrometastases can not be detected in time, and therefore we can not make sure ifthe patients have suffered from distant metastasis, so we can not make a treatmentreasonablely and immediately for all.It is because of those inevitable limitations thatthe survival of the patients with traditional treatment is limited. With the progress ofcancer biotherapy research seems to compensate for the deficiencies of traditionaltreatment. It brings hopes. CTLA-4as a target for immunotherapy of treatment ofmelanoma has achieved a major breakthrough, however, using anti-CTLA-4alone totreat breast cancer, it is no significant effect. In this study, we show the immune statusof breast cancer mouse models, and it is the first time to make and study the schemeof combination anti-CTLA-4and JAK2inhibitor to treat breast cancer in mice model.Methods:First, establish a mouse model of breast cancer subcutaneously, under the sameconditions without making any deal with mice as control, after implantation for21days, dissected mouse spleen, and the single-cell suspensions were sorted by flowcytometry assay. The percentage of CD4+、CD8+T cell, the percentage of MDSC andTreg in mice spleen，and the percentage of MDSC in mice bone marrow weremeasured.Next, following the same conditions with the aforementioned model to establishtumors, the mice were subdivided into five groups: saline control, vehicle control,anti-CTLA-4, JAK2inhibitor and combination therapy. At the third day afterinoculating we began to medicate until the day21. Then the mice-bearing tumor werekilled and the cells of spleen and bone marrow were sorted by flow cytometry cellsubtypes, using immunohistochemistry to detect the apoptotic cells and microvessel density in the tumor microenvironment.Results：1. The ratio of immune effective cells in spleen of mice bearing tumor aredecreased, and the percentage of immune suppressive cells are increased.1.1. The proportion of CD8+T cells in normal mice spleen lymphocytes is22.49±1.07, In the mice-bearing tumor, the CD8+T cells in spleen lymphocytes cells is14.74±1.86. Compared with normal mice, the percentage of CD8+T cells in spleenof mice-bearing tumor is decreased significantly (P<0.01).1.2. The share of Treg in CD4+T cell of normal mice spleen cells is13.73±1.12,in the spleen of mice bearing tumor it is27.43±0.99, the ratio of Treg in CD4+T cellof mice bearing tumor is higher than the normal mice(P<0.001).1.3. Normal mice, the ratio of MDSC in spleen mononuclear cells but notlymphocytes is11.59±3.03, the percentage of MDSC in bone marrow mononuclearcells is19.71±2.73. About mice bearing tumor, the ratio of MDSC in spleenmononuclear cells but not lymphocytes is87.50±1.60, the percentage of MDSC inbone marrow cells is94.69±0.82. Compared to normal mice, whatever in spleen orin bone marrow, the percentage of MDSC of mice bearing tumor is significantlyhigher (P<0.001).2. After treatment, the speed of tumor growth、the ratios of immune cells andsome indexes in tumor microenvironment.2.1. After combination of anti-CTLA-4and JAK2inhibitor treatment, the growthrate of the tumors in models was significantly lower than the others (P<0.05).2.2. In the group of the combination of anti-CTLA-4and JAK2inhibitor, thepercentage of CD8+T cells in spleen lymphocytes is significantly increased thanother mice bearing tumor (P<0.05). But whatever in the spleen or in the bone marrow,there is no difference about the ratio of MDSC among the groups.2.3. Compared with the control group, using anti-CTLA-4or JAK2inhibitorsalone, the ratio of apoptosis is significantly increased in the tumormicroenvironment(P<0.001). Using anti-CTLA-4and JAK2inhibitor, the number ofapoptotic cells in the tumor microenvironment is significantly higher than either of thesingle therapy group (P<0.05). After treatment of anti-CTLA-4and JAK2inhibitor,the microvessel density in tumor microenvironment is obvious decreased thanothers(P＜0.001). Using anti-CTLA-4or JAK2inhibitor alone, the microvessel density in tumor microenvironment is significantly higher than the both controlgroups(P＜0.001).Conclusions：1. In the mice bearing tumor, the percentage of cytotoxic T cells in spleenlymphocytes is significantly reduced, and the ratio of immune suppressive cells（MDSC，Treg） was obvious increased, therefore the capacity of adaptive immunesystem in mice bearing tumor is weakened.2. Combination of anti-CTLA-4and JAK2inhibitor to treat mice bearing tumor,the tumor growth is obviously limited. Apoptosis of tumor cells is increased and themicrovessel density is significantly decreased in the tumor microenvironment.