| Background:Breast cancer is the most common malignant tumor in women.Due to the continuous improvement of early diagnosis technology and treatment methods,the mortality rate of breast cancer has been declining in the past 10 years,but it is still a worldwide public health problem so far.Among breast cancers,triple-negative breast cancer and HER2-positive breast cancer are highly heterogeneous,aggressive and complex biological features,and have a poor prognosis.Now the treatment of breast cancer with a high degree of malignancy mainly relies on conventional surgery,chemotherapy and radiotherapy,but the treatment effect is still not satisfactory Therefore,looking for new breast cancer treatment strategies is of great significance to the basic research and clinical treatment of breast cancer.In 21st century,biological immunotherapy has gradually become an important means of comprehensive tumor treatment.At first,some studies found that when tumor patients were simultaneously infected with certain pathogenic microorganisms,tumor growth could be effectively inhibited and the life span of cancer patients could be prolonged.Follow-up studies have found that these infections can stimulate the host to produce multiple immune responses,and these immune responses also have anti-tumor effects while eliminating pathogens.These findings point a new development direction for cancer treatment Malaria is an intracellular parasitic infectious disease.In recent years,some researchers have found that the global incidence of malaria is negatively correlated with cancer mortality.The possible mechanism is that malaria infection activates the body’s innate and adaptive immunity,thereby improving the ability of the immune system to kill tumor cells.At present,there is little research on the anti-tumor immune mechanism of Plasmodium.The tumor microenvironment(TME)plays an important role in controlling the immune response to tumors and has become a hot spot in tumor research in recent years.Tumor-associated macrophages(TAMs)are macrophages derived from the infiltration of peripheral blood mononuclear cells into solid tumor tissues.They are part of TME and account for a large proportion of tumor stromal cells and contribute to the development of tumors.A large number of literatures show that a large number of TAMs infiltration is closely related to the poor prognosis of tumors.Studies have shown that TAMs can stimulate tumor cell proliferation,metastasis and angiogenesis,resist the killing of tumor cells by T cells and NK cells,and reduce the therapeutic effect of chemotherapy drugs.So,can Plasmodium infection lead to anti-breast cancer effect on the mouse model of breast cancer?Could the anti-breast cancer effect of Plasmodium infection be achieved by reducing TAMs recruitment in tumor tissues,thereby reducing TAMs-mediated tumor immunosuppression and thereby increasing the proportion and function of anti-tumor effector cells in TME?All these need to be further studied.Objective:1.To explore whether Plasmodium infection lead to anti-breast cancer effect on the mouse model of breast cancer.2.To explore the immune mechanism of Plasmodium infection against breast cancer from the perspective of tumor microenvironment in mouse.Methods:1.To establish a mouse model of breast cancer and carry out Plasmodium yoelii infection:40 Balb/c mice were injected subcutaneously into one side of the armpit with 1×106 4T1 breast cancer cells,and they were randomly divided into two groups with 20 in each group.One group of mice was intraperitoneally injected with 2×105 erythrocytes infected with malaria parasites on the day of modeling,and was set as the experimental(4T1+Py)group,and the other group of mice was intraperitoneally injected with the same number of normal mouse erythrocytes and set as Control(4T1)group.The tumor volume was measured every other day from the day when all the mice had tumors.The tumors were grown on the 35th day.The subcutaneous tumor tissues of half of the mice in each group were randomly stripped and weighed,and the lung tissues were stripped to count the lung metastasis nodules,and the lung tissues were stained with HE to observe the tumor metastasis.The remaining mice continue to be raised,and the survival time of each mouse is observed and recorded2.The experimental(4T1+Py)group and the control(4T1)group were also established,with 20 animals in each group.On the 28th day,the mouse tumor tissue was stripped.Immunofluorescence technology is used to label mouse breast cancer tissues with Ki-67 and CD31 fluorescence to detect tumor cell proliferation and angiogenesis3.Immunofluorescence technique was used to detect the fluorescent expression of TAMs,M2 macrophages and CD8+T cell surface antigen markers in tumor tissues4.After the tumor tissue stripped off on the 28th day was digested into a single-cell suspension by the tissue dissociation kit,the cell components(mainly tumor cells and immune cells)in the tumor tissue were concentrated by density gradient centrifugation with 40%Percoll separation solution Then,the immune cells in the tumor tissue were concentrated by the mouse lymphocyte separation solution,and the proportion of TAMs,M1,M2 macrophages,CD8+T cells and CD4+T cells were detected by flow cytometry5.The tumor tissue removed on the 28th day was used to detect the expression of CCL2,M-CSF,GM-CSF,and VEGF genes in the tumor tissue by qRT-PCR6.Magnetic bead sorting:MACS method(Militenyi Biotec)was used to purify TAMs and CD8+T cells from tumor cell suspension.The purified TAMs were tested for gene expression of IL-10,TGF-β,Arg-1,IDO,CCL-17,CCL-22,VEGFA,MMP-9 by qRT-PCR.The purified CD8+T cells were tested for the expression of IL-2,IFN-y,TNF-α,Prf and GzmB mRAN by qRT-PCR.Results:1.Compared with the 4T1 group,the tumor growth rate of the 4T1+Py group was significantly slower(P<0.001).Weighing the tumor tissue removed on the 35th day showed that the tumors of the mice in the 4T1+Py group were significantly lighter than those in the 4T1 group(P<0.001).The number of lung metastasis nodules in the lung tissue removed on the 35th day was counted,and the number of lung metastasis nodules in the 4T1+Py group was significantly less than that in the 4T1 group(P<0.001).The results of HE staining of lung tissue showed that there were more tumor metastases in the lung tissue of mice in the 4T1 group than in the 4T1+Py group.Compared with the 4T1 group,the survival time of mice in the 4T1+Py group was significantly prolonged(P<0.001)2.The immunofluorescence results showed that compared with the 4T1 group,the tumor proliferation(P<0.05)and angiogenesis(P<0.001)of the mice in the 4T1+Py group were significantly reduced3.The results of flow cytometry and immunofluorescence showed that compared with the 4T1 group,the proportion of TAMs in the tumor tissue of the 4T1+Py group was significantly reduced(P<0.05),and the polarization of M2 macrophages was also significantly down-regulated(P<0.01),the polarization of M1 macrophages did not change significantly(P>0.05),on the contrary,the proportion of CD8+T cells increased(P<0.01),the polarization of CD4+T cells did not change significantly(P>0.05)4.Compared with the 4T1 group,the expression levels of CCL2,M-CSF,GM-CSF and VEGF genes in the tumor tissues of mice in the 4T1+Py group were down-regulated(P all<0.05)5.The expression levels of IL-10,TGF-γ,Arg-1,CCL-17,CCL-22,VEGFA and MMP-9 genes in tumor tissues of mice in the 4T1+Py group were significantly down-regulated compared with the 4T1 group(P All<0.05).However,there was no significant difference in IDO gene expression level of TAMs between the two groups(P>0.05).The expression levels of IL-2,IFN-γ,TNF-α,Prf and GzmB genes in CD8+T cells in the tumor tissues of mice in the 4T1+Py group were significantly up-regulated compared with those in the 4T1 group(P all<0.05)Conclusions:1.Plasmodium yoelii infection can significantly inhibit the growth and metastasis of breast cancer in mice,and can prolong the survival time of tumor-bearing mice2.Plasmodium yoelii infection can reduce the proportion of TAMs in the mouse tumor microenvironment and inhibit their polarization to M2 macrophages,thereby promoting the proliferation of CD8+T cells and enhancing their anti-tumor immune effects3.Plasmodium yoelii infection may also reduce the microvessel density in breast cancer by down-regulating the gene expression of MMP-9 and VEGFA in TAMs to inhibiting the growth and metastasis of breast cancer in mice. |
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