The co-expression of OPN and M2-TAMs promotes gastric tumorigenesis. (a) The expression of M1/M2 markers in U937 cells was analyzed after co-culture treatment by real-time PCR. LPS-treated U937 cells expressed IL-1 as M1 macrophages, and U937 cells alone were used as a negative control. TAMcli cells isolated from clinical specimens were used as a positive control. After co-culture with OPN+-AGS cells, the U937 cells differentiated into M2-TAMs expressing high levels of CD204 and IL-10. (b) After 72 hours of co-culture with U937 or TAMcli cells, the invasiveness of the OPN+-AGS cells increased but that of OPN-shRNA AGS cells did not. The increased cell invasiveness was also blocked by an OPN monoclonal antibody. (c) The mixture of co-cultured OPN+-AGS and U937 cells inoculated into nude mice showed poor survival compared with a mixture of co-cultured OPN-shRNA AGS and U937 cells or OPN+-AGS cells alone (p = 0.0498). (d) The neovascularization in the xenografts was shown by double-staining with anti-CD31 and anti-αSMA antibodies. Less neovascularization with normal vascular structure was found in xenografts of a mixture of co-cultured OPN-shRNA AGS and U937 cells.