A Study On The Polarization Of Macrophages Treated With The Conditioned Medium

Macrophages are a major cellular component of tumor microenvironment. Upon the stimulation of cytokines and secretory proteins in tumor microenvironment, macrophages can be switched from M1 macrophages to M2 macrophages. It is generally accepted that M1 macrophages are involved in the inflammatory response and antitumor immunity, but M2 macrophages exert anti-inflammatory and pro-tumorigenic activities. The macrophage polarization is regulated by environmental components derived from tumor and stromal cells, such as cytokines, chemokines and extracellular matrix (ECM).Our study focused on cytokines and molecules derived from tumor cells and intended to explore their functions on macrophages polarization in tumor microenvironment. We constructed NIH3T3/Src cells which is stably overexpressing Src protein, and found its conditioned medium induced ANA-1 cells to M2-like phenotypes. To identify the specific components of NIH3T3/Src cells conditioned medium which induced the polarization of macrophages, we tested the cytokines in NIH3T3/Src cells conditioned medium by cytokines antibody array. The results showed higher concentration of IL-6, MCP-l,sTNFR, VEGF, KC, GM-CSF and axl in NIH3T3/Src cells conditioned medium compared with control. As these proteins were regulated by NF-κB and JAK-STAT signal pathways, we inhibited NF-κB and JAK-STAT signal pathways by their specific inhibitors PDTC and AG490. The results of ELISA showed inhibition of IL-6 after inhibition. Meanwhile, the switch of macrophages to M2-like phenotypes induced by conditioned medium decreased significantly. These results indicated that IL-6 derived from NIH3T3/Src cells plays a key role in macrophages polarization. In addition, Lactic acid was also accumulated in the supernatant of NIH3T3/Src cells. Inhibition of lactic acid intake by macrophages by a cyano-4-hydroxy cinnamic acid (CHC) resulted in decrease of the macrophage polarization induced by NIH3T3/Src cells conditioned medium, which indicated that lactic acid involved in the polarization of macrophages.With the development of colorectal cancer in C57BL/6J-ApcMin/+ mice, the secretion of factors such as IL-6, IL-9 and IL-10 in interstitial fluid of mice colorectal tissues increased. In IL-6tmlKoPf mice (IL-6 knockout), when used AOM-DSS to induce mice colorectal cancer, the tumorigenesis in IL-6tmlkopf mice was obviously inhibited compared with control group, which suggested the important roles of IL-6 in tumorigenicity.In summary, IL-6 and lactic acid secreted from NIH3T3/Src cells play important roles in the polarization of macrophages. While further investigation is warranted, our present study reveals a possible mechanism of macrophage polarization induced by conditioned medium. Moreover, it implies a potential strategy to inhibit macrophage polarization, which is a promising novel method for cancer therapy.