Fig. 4.

Tumor cells and tumor-secreted factors stimulate TNF-α production by monocytes. a Bone marrow monocytes were cultured overnight with B16 cells or 4T1 cells, or with tumor CM derived from these cells. Medium from monocyte cultures was collected 24 h after co-culture with live tumor cells or with tumor CM, and IFN-γ and TNF-α concentrations were determined by ELISA. b Bone marrow-derived monocytes were cultured overnight with tumor CM then incubated with Brefeldin A for 4 h and immunostained for detection of intracellular TNF-α expression by flow cytometry. Appropriate isotype control antibodies were used to assess the specificity of TNF-α staining. Similar results were obtained in three additional independent experiments. Statistical comparison of TNF-α concentrations was done by one-tailed ANOVA, with Dunnet’s post-test. Statistically significant differences were denoted as *** = p < 0.0005 and **** = p < 0.0001