Application Of IPhoneDB To Explore The Interaction Of Breast Tumor Cells Between Macrophages Under Chronic Stress-induced Epinephrine Treatment

1.Research backgroundThe chronic stress caused by modern life is closely related to the occurrence and development of tumors.Chronic stress causes the release of chronic stress-related hormones such as epinephrine,norepinephrine,and cortisol by changing the signal pathway of the hypothalamus-pituitary-adrenal axis and the sympathetic nervous system,thus affecting tumor progression.Research has found that these hormones affect tumor proliferation,angiogenesis,immune escape,genome stability,metastasis,and metabolic abnormalities.At present,the research mainly focuses on the effect of hormones produced by chronic stress on tumor cells,but the interaction between tumor cells and immune cells in tumor microenvironment under this condition needs further study.The tumor microenvironment is infiltrated with a large number of immune cells,including antigen presenting cells,T cells,B cells,dendritic cells,myeloid-derived suppressor cells,macrophages,etc.The immune cells and their secreted cytokines are important components of the tumor immune microenvironment.Intercellular interaction is one of the research hotspots in recent years.There is a ligand-receptor pair based interaction between immune cells and tumor cells.However,under the stimulation of epinephrine generated by chronic stress,the interaction between tumor cells and immune cells is rarely reported.Our research group has developed a database iPhoneDB for the study of cell-cell interaction.IPhoneDB integrates the prior knowledge of ligand-receptor interaction and models the communication probability between cells,including the interaction between immune cells and tumor cells.On the basis of inferring the intercellular communication network,it provides a research platform for further data exploration,analysis and visualization.Specifically,we can predict which ligands in one cell population("sender")may affect which receptors in another cell population("receiver")through the interaction of ligand receptor pairs,and we can also infer the possible interaction between two specific cell types through cell markers.Therefore,this topic attempts to apply iPhoneDB to explore the interaction of epinephrine generated by chronic stress on breast cancer cells and macrophages.2.Research methods(1)① Establish the research strategy of "tumor cell-immune cell" interaction:treat breast cancer cell MDA-MB-231 with DMSO and epinephrine in vitro,and carry out microarray detection to obtain differentially expressed genes(DEG);②The differentially expressed genes and iPhoneDB were analyzed jointly to screen the potential interaction molecules between tumor cells and immune cells.After the treatment of DMSO and epi selected above,there were differentially expressed tumor cell ligands and receptors that might participate in their interaction with immune cells.(2)①MCF7 and MDA-MB-231 cells were treated with DMSO and epi.During 2D culture,the medium condition was DMEM added with 2%fetal bovine serum and 10 nM epinephrine for 5 days.During 3D culture,the cells cultured in 2D for 5 days were collected and inoculated to the low adhesion cell culture plate.The medium condition was sphere culture medium added with 10 nM adrenaline,and 500 μ L medium were replenished every other day for 7 days;②Collect MCF7 and MDA-MB-231 cells treated with DMSO and epi in 2D and 3D culture for WB or QPCR experiments;③ According to iPhoneDB,receptors on macrophages are speculated.(3)① Add the inducer Phorbal-12-myristate-13-acetate(PMA)to the monocyte THP-1 for 2 days,observe the cell adhesion,remove the PMA,and culture for 3 days at rest,collect the cells for flow cytometry detection;② The morphology of monocyte THP-1 after PMA treatment was observed under microscope.(4)① MDA-MB-231 cells were treated with epinephrine,and the basic biological functions were tested,including CCK8 test,PY-hochest staining,and cell activity staining;② After the monocytes were induced to adhere to the plate by PMA,the PMA was removed,and the culture medium was changed to 1640 with 2%fetal bovine serum and 10 nM epinephrine.After treatment for 3-5 days,the cells were collected for CCK8 test and flow cytometry detection.(5)① Collect the culture medium of macrophages and tumor cells treated with DMSO and epi for 3 days;②The co-culture system was designed,and the transwell experiment was adopted.When the cells were cultured separately,the ordinary medium containing DMSO or epi was placed under the transwell,and the corresponding treated macrophages were placed in the transwell.When the cells were co-cultured,the tumor cell culture medium treated with DMSO or epi was placed under the transwell,and the corresponding treated macrophages were placed in the transwell.After 6 hours of culture,the transwell was turned over,and the cells were stained by hochest and photographed by fluorescence microscope.3.Research results(1)① Analyze the microarray data of tumor cells treated with DMSO and epi,and obtain 82 up-regulated and 101 down-regulated differentially expressed genes under the conditions of P value<0.05 and Fold change>2;②The differentially expressed genes were intersected with iPhoneDB to obtain the differentially expressed ligand genes COL15A1,ENPP2,SLPI,ADM,PLAT,LAMB3,CSF2,TNC,PRSS3 and receptor genes TGFBR1,F3,SDC2,TNFRSF21 on tumor cells.(2)① WB showed that the expression of ADRB2 in tumor cells was up-regulated after epinephrine treatment,indicating that the model of epinephrine treatment in vitro was successfully constructed;②The QPCR results of 2D and 3D cultured cells showed that PLAT and CSF2 were consistent with the expression trend of ligand and receptor in the joint analysis;③ According to the principle of ligand-receptor pairing to mediate the interaction between cells,PLAT can be paired with receptors ITGB2 and ITGAM on macrophages,and CSF2 can be paired with receptors CSF1R on macrophages,suggesting that epinephrine treatment may affect the interaction between tumor cells and macrophages,and promote the recruitment of tumor cells to macrophages.(3)① Microscopic observation showed that monocytes began to adhere to the plate after PMA treatment;② Flow cytometry showed that when PMA was not added,the positive rate of CD11b was 15.00%,and when PM A concentration was 50,150,and 300 nM,the positive rate of CD11b was 65.52%,66.59%,and 73.59%,respectively,indicating that when PMA concentration was 300 nM,it could better induce monocytes to form macrophages.(4)① CCK8 experiment showed that epinephrine had no significant effect on the proliferation of MDA-MB-231;② PY-hochest flow cytometry showed that epinephrine had no significant effect on the cell cycle of MDA-MB-231;③FVD dye flow cytometry showed that epinephrine had no significant effect on the cell activity of MDA-MB-231;④CCK8 experiment showed that epinephrine had no significant effect on the proliferation of macrophages;⑤CD11b flow cytometry showed that epinephrine had no significant effect on differentiation of macrophages.(5)① Fluorescence microscope observation showed that there was no significant difference in the number of macrophages passing through the transwell when cultured alone,which proved that there was no significant effect on the migration of macrophages with or without epinephrine treatment.When co-cultured,the number of macrophages passing through the transwell in the epinephrine treatment group increased significantly,which proved that the tumor cell culture medium after epinephrine treatment would promote the migration of macrophages.4.Research conclusions(1)Application of DEG and iPhoneDB combination analysis to screen the interaction molecules between breast tumor cells and macrophages under chronic stress-induced epinephrine treatment;(2)Epinephrine produced by chronic stress regulates the abnormal expression of ligands PLAT and CSF2 in tumor cells that interact with macrophages;(3)In mono-culture system,it was observed that epinephrine produced by chronic stress had no significant effect on the proliferation,cell cycle,activity of breast cancer cells and the proliferation and differentiation of macrophages;(4)In co-culture system,epinephrine produced by chronic stress promoted breast cancer cells to recruit macrophages.