Proteomic Analysis Of Colorectal Tumor-educated Macrophages

The crosstalk between the tumor cells and the surrounding cells includingmacrophages, fibroblasts and extracellular matrix forms tumor microenvironment,promoting proliferation, invasion and metastasis of cancer cells. Tumor-associatedmacrophages are a key player in this process.Aim:We aimed to explore the proteomic differences between cancer cell-educatedmacrophages (CEM) and normal macrophages (Mφ), to probe the signaling pathwaysrelated to this phenotypic transformation.Methods:Supernatants of Caco-2cell cultures were employed to treat THP-1macrophagesto establish a CEM model. Proteomic differences between Mφ and CEM wereanalyzed by two-dimensional electrophoresis (2-DE) and matrix-assisted laserdesorption ionization time of flight tandem mass spectrometry(MALDI-TOF/TOF-MS). Ingenuity pathways analysis (IPA) software was used toanalyze the biological pathways involved by the altered proteins. Western blotting andfluorescence microscope were used to validate the biological functions of the proteins.Supernatants of CT26cell cultures were employed to educate bonemarrow-derived macrophages to establish another CEM model. SILAC technique,LC-MS/MS analysis, IPA and biological validation were performed to characterize theproteomic differences between the CEM and the normal macrophages.Results:A total of60differentially expressed proteins (DEPs)(Cut off≥2-folds) wereidentified in THP-1CEM. Among them,13proteins were upregulated and47weredown-regulated. IPA analysis indicated that these DEPs were involved in functionalchanges in apoptosis pathways, specifically showing an anti-apoptotic phenotype inCEM. This speculation was experimentally verified by the upregulation of Bcl-2andthe downregulation of cleaved caspases3&7in CEM.In total1296proteins with more than2unique peptides were identified by LC-MS/MS in CEM from bone marrow-derived macrophages. Among them,58proteins were upregulated and42were down-regulated (Cut off≥1.5-folds). IPAanalysis suggested that these DEPs were involved in the functional changes in lipidmetabolism, molecular transport and protein trafficking pathways. WST-1testindicated that the proliferative capacity of CEM was significantly enhanced ascompared to the normal macrophages.Conclusions:We successfully constructed in vitro models for studying the tumor-associatedmacrophages and used these models to quantitatively analyze the proteomicdifferences between normal macrophages and the CEMs.Cancer cells may stimulate the anti-apoptotic and proliferation-promotingpathways in CEMs.