Metastasizing cancer cell-derived factors induced excessive IL-6 production by MDSCs. (A) Splenic MDSCs (4×105) were co-cultured with 4T1 cells (1×104) or EMT6 cells (1×104) for 48 hours. IL-6 levels in the culture supernatants were measured by ELISA. (B-C) Splenic MDSCs from 4T1 cell-bearing mice were co-cultured with 4T1 cells in Transwell systems (B) or exposed to conditioned media (CM) for 24 hours (C). (D) Splenic MDSCs were cultured with 4T1-CM in the presence of signaling inhibitors for 24 hours. (E) Immunofluorescence staining of Gr-1 (red), IL-6 (green), and DAPI (blue) in the spleen, tumors and lungs of 4T1 cell-bearing mice. Scale bar = 30 μm (original magnification, ×1,000). MDSCs, myeloid-derived suppressor cells.