Effects Of 4T1 Breast Cancer Cell Microenvironment On The Proliferation And Differentiation Of RAW264.7 Macrophages

Among the most common malignant tumors with women in China,breast cancer occupies an important position.Around the world,the incidence and mortality of breast cancer are gradually showing a younger and increasing trend.It is well known that macrophages are important immune cells in the body’s innate immunity.Recent studies have shown that tumor tissues are infiltrated with a large number of differentiated macrophages,which are called tumor-associated macrophages(TAMs),especially in breast cancer tissues.A large number of evidences have shown that TAMs do not play an anti-tumor role by inducing immune response in the occurrence,development and metastasis of breast cancer,but play a role in promoting tumor progression.At present,it is believed that there are two polarized states of TAMs:classic activated type(M1)and alternative activated type(M2).M1 TAMs play pro-inflammatory and anti-tumor roles in the occurrence and development of breast cancer,while M2 TAMs play a promoting role in breast cancer immunosuppression,proliferation and drug resistance.The polarization pattern of TAMs changes with the different tumor microenvironment.TAMs are important immune cells in the breast cancer microenvironment,and breast cancer cells also depend on the breast cancer microenvironment to survive.In recent years,more and more studies have suggested that the breast cancer microenvironment drives the polarization of tumor-associated macrophage phenotype.Objective:In this study,the culture supernatant of mouse 4T1 breast cancer cell and the supernatant of mouse 4T1 breast cancer cell whole cell antigen were used to simulate the microenvironment of mouse breast cancer in vivo to culture RAW264.7 macrophages.To observe the changes of morphology,proliferation activity and related cytokine m RNA expression levels of mouse RAW264.7 macrophages in the tumor microenvironment of mouse 4T1breast cancer cells in vitro.To further explore the molecular mechanism of tumor-associated macrophages function changes in the breast cancer microenvironment,so as to play a positive role in anti-tumor activity of tumor-associated macrophages and provide theoretical basis for future immunotherapy of breast cancer.Methods:1 Subjects:mouse 4T1 breast cancer cells and mouse RAW264.7macrophages.2 Group of Experiments:control group:RAW264.7 macrophages were cultured in DMEM complete culture medium;4T1 breast cancer cell culture supernatant group:RAW264.7 macrophages were cultured in DMEM medium containing 25%or 50%concentration of 4T1 breast cancer cell culture supernatant,respectively.4T1 breast cancer cell whole cell antigen group:RAW264.7 macrophages were cultured in DMEM medium containing10%or 20%concentration of 4T1 breast cancer cell whole cell antigen supernatant,respectively.3 Preparation of 4T1 breast cancer cell culture supernatant:4T1 breast cancer cells were inoculated in T25 culture flask containing 1640 culture medium at a concentration of 2.5×10~5/ml.After 72 hours of culture,the supernatant was collected by centrifugation and filtration to remove bacteria,store at-20℃until use.4 Preparation of 4T1 breast cancer cell whole cell antigen supernatant:4T1 breast cancer cells were packaged in frozen tubes at a concentration of5×10~6/ml,repeatedly frozen and thawed in liquid nitrogen for 3 times,and then the supernatant was collected,centrifuged and filtered to remove bacteria,store at-20℃until use.5 The morphology and changes of RAW264.7 macrophages under inverted microscope on the 3rd and 5th day of culture in each experimental group.6 MTS experiment was used to detect the proliferation of RAW264.7macrophages on the 3rd and 5th day of culture in each experimental group.7 Real-time PCR experiment was used to detect the m RNA expression of cytokines such as interleukin-6,interleukin-10,interleukin-12,tumor necrosis factor-αand other cytokines in RAW264.7 macrophages on the 3rd and 5th day of culture in each experimental group.8 Statistical methods:SPSS 27.0 was used to analyze the experimental data.Results:1 The morphology of RAW264.7 macrophages was observed under inverted microscope:Compared with the control group,the RAW264.7macrophages in the culture supernatant of 4T1 breast cancer cells grew faster.On the second day of culture,the volume of macrophages became larger and dendritic changes appeared,which were more significant with the increase of the supernatant concentration.There was no significant difference in RAW264.7 macrophages between 4T1 breast cancer cell whole cell antigen group and control group.2 The results of MTS experiments showed that compared with the control group,the proliferation rate of RAW264.7 macrophages in the 4T1breast cancer cell culture supernatant group was significantly increased after3 days of culture(P<0.001),and the proliferation rate was significantly decreased after 5 days of culture(P<0.001).The proliferation rate of RAW264.7 macrophages in 4T1 breast cancer cell whole cell antigen group increased first and then decreased with the increase of antigen concentration on 3 and 5 days of culture.However,the overall pattern of change was still increasing compared to the control group(P<0.05).3 The results of Real-time PCR experiments showed that:In contrast to the control group,after 3 days of culture,the m RNA expression levels of IL-6,IL-12 and TNF-αin RAW264.7 macrophages in 4T1 breast cancer cell culture supernatant group and whole cell antigen group were decreased(P<0.05),and the m RNA expression level of IL-10 in supernatant group was elevated(P<0.05).However,the expression level of IL-10 in antigen group was not significantly increased.After 5 days of culture,the m RNA expression levels of IL-10 and IL-12 in the supernatant group increased(P<0.05),the expression level of IL-6 decreased(P<0.05),and the expression level of TNF-αdecreased first and then increased with the increase of the concentration of supernatant(P<0.05).Interestingly,the levels of IL-6,IL-10,and IL-12 m RNA expression in the antigen-treated group were significantly lower than those in the control group(P<0.05),and the expression level of TNF-αwas not significantly changed.Conclusions:1 The tumor microenvironment simulated by 4T1 breast cancer cell culture supernatant can activate the proliferation of RAW264.7 macrophages,promote the proliferation of cells,and induce the polarization of RAW264.7macrophages to M2 macrophages and its subtypes.2 The tumor microenvironment simulated by 4T1 breast cancer cell whole cell antigen promoted the proliferation of RAW264.7 macrophages at low concentration,but inhibited the proliferation of RAW264.7 macrophages at high concentration.To a certain extent,the tumor microenvironment could also induce the polarization of RAW264.7 macrophages to M2 macrophages at early stage.