The paracrine effect of OPN skewed macrophages toward M2-TAMs in gastric cancer. (a) Dual immunofluorescence staining of OPN (green) and CD204 (red) shows co-localization (yellow) in gastric cancer specimens. The immunohistochemistry of CD204 shows that macrophages did not come in contact with tumor cells. (b) A co-culture method was designed to mimic the tumor microenvironment of gastric cancer without direct contact between cancer cells and macrophages. (c) The confocal images show that OPN contributed to the recruitment of U937 cells and skewed the cells toward M2-TAMs after treatment with conditioning medium; this phenomenon could be blocked by an OPN monoclonal antibody. Recombinant OPN was able to increase CD204 expression on macrophages. (d) The amount of CD204+TAMs was shown by flow cytometry. The CD204+-TAM phenotype increased dramatically after co-culture with OPN+AGS cells (red, open histogram). Parental cells (filled histogram), OPN-shRNA AGS cells (orange, open histogram), OPN neutralizing antibody (green, open histogram), or recombinant OPN (blue, open histogram) were compared. The TAMcli cells isolated from human gastric cancer were used as a positive control.