Effect Of Macrophages On Gastric Cancer Stem Cells In Co Culture System

Background:The milky spot is a structure which gathering a large amount of macrophages, it has important immune functions in tumor development, invasion and metastasis,especially in the function of angiogenesis.We had found that gastric cancer cell selectively invade milky spots when gastric cancer appear metastasis at peritoneal in our previous studies. Most of gastric cancer cells invaded the milky spots were destroyed by macrophages. But a small part of cells survived in the milky spots can be to increase in the environment of macrophages.Objective:We consider that the part of residual cells are cancer stem cells(CSCs) which reserve ability of replication and can escape from immune cell. We design the co-culture system by gastric cancer stem cells and tumor associated macrophages to explain this phenomenon in this experiment. We try to explore the changes of gastric cancer stem cells which in the milky spot by the simulation of micro environment under the co-culture system.Methods:First we pick up the cell sphere from the SGC-7901 cell culture, to accomplish the purpose of isolating and transfering of culture of the gastric cancer stem cells,. In the co-culture system we established the detection of gastric cancer stem cell survival rate by MTT method. We detect the changes of the clone ability and study the expression in Caspase-3/9、IL-4,IL-10、INF-γ、IL-12、MMP-2、MMP-9、 MCP-1、COX-2、PGE2、VEGF、TGF-β of gastric cancer stem cell byRT-PCR, Western blot, ELISA.Results:The viability of gastric cancer stem cell increased in this co-culture system with macrophages concentration of 2 x 105 /ml, 4 x 105 /ml.The clone forming efficiency(CFE) was(0.811±0.035) % in 0 cells of macrophages 、(0.612±0.157) % in 1 x 105 cells of macrophages、(0.4057 ± 0.016) % in 2 x 105 cells of macrophages、(0.213± 0.413) % in 4 x 105 cells of macrophages,and the difference is statistically significant(P < 0.01). The expression level of Caspase-3/9 of gastric cancer stem cells increased with the concentration of macrophages were cultured in vitro,and the activation of apoptosis of gastric cancer stem cell.was picked up under this system MCP-1 has an important role on tumor growth, invasion and angiogenesis as the main chemokine. In the co-culture with macrophages and gastric cancer stem cells activity of MCP-1 was enhanced, it showed that stem cells may be activated in the inflammatory environment COX-2 is a rate-limiting enzyme of arachidonic acid with a variety pathway to resist apoptosis of tumor, and it can reduce level of transforming growth factor TGF-β and modify apoptosis promoting tumor growth. The COX-2 was expressed high under the co-culture system and showed higher ability anti apoptosis with different levels of macrophage. In co-cultured with gastric cancer stem cells and macrophage TGF-β in the low expression of COX-2 downstream the expression in mRNA and protein in inflammatory environment. The mRNA and protein activity of PGE2, high expressed in the co-culture to inhibit apoptosis of gastric cancer stem cells. The expression of mRNA,protein of VEGF was activated by macrophages with concentration of 1 x 105, 2 x 105 and 4 x 105 /ml. Tumor cells and tumor associated macrophages can secrete immunosuppressive factor IL-10,IL-10 can promote macrophage differentiation of tumor type M2 macrophages, inhibite the inflammatory reaction, promote angiogenesis and tissue repair. IL-10 activity of gastric cancer stem cells decreased In co-cultured with macrophages under the environment. IL-4 high express in serum of gastric cancer patients, IL-4 and IL-10 are also highly expressed in M2 macrophages, cells in co-culture with macrophages The expression of IL-4 in gastric cancer stem cell was inhibited. M1 macrophages high expressed IL-12 on inhibiting the growth and metastasis of the tumor. IL-12 was high expressied in the environment of gastric cancer stem cells with macrophages. In macrophage co-culture environment, although the expression of IL-4, IL-10 and IL-12 of gastric cancer stem cells showed bidirectional, but the activity of gastric cancer stem cell was inhibited by macrophage. The high expression of IFN- γshowed activity gastric cancer stem cell was inhibited by macrophage. MMP-2 and MMP-9 belong to matrix metalloproteinases can affect cell adhesion molecules, extracellular matrix degradation mediated by tumor cells of the host cells, generating regulation of tumor angiogenesis. Effect of MMP-2 and MMP-9 of gastric cancer stem cells was low expressed by macrophage cells, the invasion of stem cell was inhibited by macrophage.Conclusion:Although the MTT assay and clone forming efficiency showed that performance of gastric cancer stem cells was inhibited by macrophage in the co-culture system with gastric cancer stem cells and macrophage of inflammatory environment The reason was considered that Caspase-3/9, IL-4, IL-10 and interferon gamma was activated, IL-12, MMP-2 and matrix metalloproteinase MMP-9 was inhibited. Because that MCP-1, COX-2, PGE2, VEGF was also highly expressed and TGF-β was low expressed anti apoptosis and promoting the ability of angiogenesis.of gastric cancer stem cells were reflected. We can see that gastric cancer stem cells decrease the number of cells and reduces the ability of cloning. in the co-culture system and the immunotherapy is still important therapeutic method of gastric cancer.