Quantitative Proteomic Analysis On Tumor Associated Macrophages

Research on cancer has been lasting for more than a century. In the last decade, researchers have paid more attention on the tumor microenvironment that has been demonstrated to maintain and promote the tumor growth and metastasis. Tumor associated macrophages (TAM) are the major component of the nonmalignant cells in the tumor microenvironment. As other innate immune cells, macrophages are capable to adjust themselves according to the environmental change. Ml and M2 are the two extreme polarizations of TAMs. M1 macrophages can be induced by microbial components such as LPS or IFN-r that play critical roles in anti-infection and acute inflammation. On the contrary, M2 macrophages can be induced by IL-4 and IL-13, tending to produce anti-inflammtory and immune-suppressive factors such as IL-10 and glucocorticoid. M2 macrophages are also demonstrated to function in tissue remodeling and angiogenesis. TAMs are thought to have a M2 like phenotype, because of their capability to produce more IL-10 and their deficiency in antigen presentation. The mechanisms by which the tumor microenvironment drives macrophage polarization and TAMs promote tumorigenesis and metastasis are poorly understood.Aims:By performing SILAC-based quantitative proteomics, we aimed to unravel the signal networks that regulate macrophage polarization and to discover the TAMs-supplied proteins that play crucial roles in promoting tumor development and invasion.Methods:Using flow cytometer and ELISA to access the surface markers and cytokine pattern of the tumor-educated macrophages (TEMs). The THP-1 macrophages and the tumor-educated macrophages were labeled with light and heavy SILAC reagents, respectively. After 6-day education by tumor cell supernatant (TSN), the THP-1 macrophages and TEMs were harvested and the cellular proteins were extracted and digested by trypsin. The mixture of peptides was analyzed by LC-MS/MS. The raw data were processed by Maxquant, and the bioinformatic analysis was mainly accomplished with IPA system.Results:The M2 markers CD206, CD163, IL-10 were significantly upregulated while the M1 marker CCR7 was strikely down-regulated after the education of THP-1 macrophages with TSN, indicating that the TEMs have a M2 like phenotype.1296 proteins with more than 2 unique peptides were identified by LC-MS/MS. Among them,253 proteins were upregulated,131 were down-regulated (Cut off> 1.5-folds). IPA analysis revealed the high confident signalling networks are closely related to tumorigenesis and invasion. The cathepsin family members, CTSB, CTSD, CTSL, CTSS, were all upregulated and included in a network that highly related to tumor invasion and metastasis. These data indicated that the cathepsin family may participate in the development and metastasis of cololectal cancer.Conclusions:We successfully constructed an in vitro model for studying the tumor-associated macrophages, and used this model to quantitatively analyze the protein profile of the TEMs. The SILAC-based proteomic analysis elicited great repeatability (> 70%). Signalling networks revealed by bioinformatics implied cathepsins as the potential target of cancer therapy.