The tumor microenvironment is a complex milieu that includes not only malignant cells, but immune cells, fibroblasts, signaling molecules, the extracellular matrix (ECM), and blood vessels. We have previously found that cmvIL-10 enhances migration of MDA-MB-231 breast cancer cells in vitro toward epidermal growth factor (EGF) . In order to more faithfully replicate conditions under which cmvIL-10 might be found in the tumor microenvironment, we examined the ability of cmvIL-10 secreted from virus-infected cells to stimulate movement of MDA cells. Monolayer cultures of human foreskin fibroblasts were mock-infected or infected with HCMV strain AD169 at a range of multiplicities of infection (MOI). After 96 h, supernatants were harvested and placed in the lower chamber of a transwell migration plate in the presence or absence of EGF. MDA cells were placed in the upper chamber, separated from the EGF and conditioned medium by a porous filter. After five hours, cells that traversed the filter were quantified. MDA cells did not exhibit any significant movement toward conditioned medium from mock or infected cells, which is consistent with our previous finding that cmvIL-10 is not a chemoattractant for tumor cells . However, when conditioned medium from mock infected cells was supplemented with EGF, cell migration was observed (Fig. 1a). When EGF was added to conditioned medium from HCMV-infected cells, the amount of cell migration increased, suggesting that substances released from virus-infected cells amplified chemotaxis to EGF. Moreover, the enhanced MDA cell movement was greater when EGF was provided in supernatants from higher MOI infections, and thus greater concentrations of cmvIL-10, indicating a dose-dependent effect. To confirm that cmvIL-10 was the virally produced substance mediating this increase in cell movement, MDA cells were pre-incubated for 30 min with a neutralizing antibody (NAb) directed at the cellular IL-10R. The NAb was also included in the top chamber with MDA cells during the 5 h incubation, and resulting migration was reduced to levels seen when only EGF was present in medium from mock infected cells. These results demonstrate that cmvIL-10 secreted from virally infected cells has the ability to interact with the cellular IL-10R on tumor cells to enhance directed movement.Fig. 1MDA-MB-231 cells exhibit increased migration and matrigel invasion in the presence of cmvIL-10. a Transwell migration toward EGF after 5 h in the presence of conditioned medium from mock or HCMV-infected fibroblasts at the indicated MOIs. IL-10R neutralizing antibody (NAb) was included at 30 μg/ml. b Matrigel invasion toward EGF in the presence of 100 ng/ml purified recombinant cmvIL-10 or hIL-10 after 22 h. c Matrigel invasion toward EGF in the presence or absence of 100 ng/ml purified recombinant cmvIL-10 or conditioned medium from mock or infected fibroblasts (MOI = 1). Where indicated, 10 μM Stat3 inhibitor was included. Error bars SEM. *p < 0.05, **p < 0.001. These results are representative of three independent experiments