Stimulation of MDSCs with metastasizing 4T1 cell-derived factors induced persistent Stat3 phosphorylation in cancer cells. (A) Splenic MDSCs from 4T1 cell-bearing mice were cultivated in the presence of 4T1-CM or EMT6-CM. The conditioned media (MDSC-CM, 4T1/MDSC-CM, EMT6/MDSC-CM) were harvested and applied to 4T1 and EMT6 cancer cells. (B-C) Phospho-Stat3 and Stat3 levels in 4T1 cells exposed to each CM for 10 minutes (B) and for the indicated periods of time (C). (D) 4T1 cells (1×104) were co-cultured with splenic MDSCs (4×105) from 4T1 cell-bearing mice or recombinant mouse IL-6 (1 ng/ml). Phospho-Stat3 and Stat3 levels in 4T1 cells were determined by Western blotting after the removal of MDSCs. CM, conditioned media; MDSCs, myeloid-derived suppressor cells.