The Comparative Proteomics Study Of Mouse Macrophages And Tumor Associated Macrophages

The tumor microenvironment consists of tumor cells, and their surrounding immune cells, fibroblasts, endothelial cells, blood vessels, signaling molecules and the extracellular matrix. Tumor cells and the surrounding microenvironment are closely related and interact constantly. Tumor cells can influence the microenvironment by releasing many functional molecules through autocrine and paracrine to promote their own progression and the homeostasis status of the tumor microenvironment. Macrophages represent up to 50% of the tumor mass, and they certainly operate as fundamental actors. Tumor associated macrophages (TAMs), the major players in the connection between inflammation and cancer, which have been reported to regulate the promotion of tumor cell proliferation, angiogenesis and repression of adaptive immunity through secreting many kinds of mediators including inflammatory cytokines, proteases and growth factors. According to recent studies, TAMs could also to regulate the functions of their recipient cells through releasing exosomes, a nano-sized particle. However, the protein compositions of TAMs and derived exosomes are rarely known till now.Here, we constructed a TAMs cell model by tumor media inducement from a mouse macrophage cell line, Ana-1, and performed comparative proteomics on cells, exosomes, and exosome-free media between TAMs and Ana-1. The proteome of TAMs whole cell suggested that tumor secreted proteins greatly reprogrammed metabolism status of macrophages, such as amino acid, glucose and fatty acid metabolism. This finding suggested that in addition to regulating signaling pathway of stromal cells in tumor microenvironment, tumor cells might also regulate stromal cells by altering their metabolism status. In addition, the cellular proteomic data suggested that several upstream and downstream molecules of GR signaling pathway were up-regulated. We validated that the nuclear translocation of GR was increased in TAMs and its downstream molecules such as anti-inflammatory cytokines IL-10 and IL-1RA were also up-regulated in TAMs. The result suggested that the activation of GR signaling pathway might result in the anti-inflammatory phenotype of TAMs model in our study.Proteomic analysis between exosome and exosome-free fractions derived from Ana-1 and TAMs indicated that the functions of exosome dominant proteins were mainly enriched in RNA processing and proteolysis, while homeostatic processes related proteins were through exosome-free secretory path. Comparison of exosomes proteomes derived from mouse macrophage with those from three other types of cells strongly suggested that macrophage-derived exosomes have cell-specific potential, which mainly enriched in RNA processing and ribosome biogenesis processes. According to the proteomics data, TAMs status dramatically affected the abundances of 20S proteasome subunits and ribosomal proteins in their exosomes. The 20S proteasome activity assay strongly indicated that TAMs exosomes possessed higher proteolytic activity. In addition, Ana-1 and TAMs-derived exosomes have different RNA profiles, which might result from differential RNA binding proteins. These results suggested that exosome released by TAMs might have potential to regulate tumor progression through degrading misfolded proteins or delivering distinct RNAs in the recipient tumor cells.Taken together, through comprehensive proteomics study on the whole cellular proteome and secretome including exosome and exosome-free derived from Ana-1 and TAMs, and subsequent functional analysis, we identified unique features of TAMs and its exosomes. Our study provides novel views for understanding the complicated roles of TAMs, especially TAMs-derived exosomes in tumor microenvironment.