Tumor growth inhibition by activated M1-like macrophages is mediated by soluble factors. (A) HOS-143b cells were incubated with M1-like macrophages (pre-activated +/− LPS + IFN-γ or L-MTP-PE + IFN-γ) (no pattern) or with cell culture supernatant from these macrophages (hatched pattern) for two days. Tumor cell numbers were analyzed relative to the control (ANOVA and Dunnett’s post test n = 3–8). The cytokine/chemokine profile of M1-like macrophages was assessed in cell-free supernatants obtained (B) after macrophage activation or (C) after macrophage activation and subsequent two-day co-culture with HOS-143b cells. Data of IL-1β, IL6, IL-12p70, TNF-α, CXCL10 (IP-10), CCL2 (MCP-1), CCL3 (MIP-1α), CCL4 (MIP-1β) and CCL5 (Rantes) were acquired by Luminex assays (n = 2–3). There was no cytokine/chemokine production by tumor cells alone. Compared to activated macrophages alone the co-culture with tumor cells did not enhance cytokine/chemokine production (data not shown). (D) HOS-143b cells were incubated with/without LPS + IFN-γ-activated M1-like macrophages in the presence or absence of inhibitors against TNF-α (by neutralizing antibody and soluble TNF receptor), IL-1 receptor (by IL-1Ra), TNF-α and IL-1 receptor, nitric oxide (by L-NAME) or reactive oxygen species (by catalase and SOD). For each set of inhibitor experiments (n = 3–6) tumor cell numbers of the following conditions are depicted: after co-culture with activated macrophages (filled bar), tumor cells alone with inhibitors (white bar hatched pattern), after co-culture with activated macrophages with inhibitors (filled bar hatched pattern), analyzed relative to tumor cells only without inhibitors (white bar). There was no significant difference between co-cultures with and without inhibitors, or between tumor cells alone and co-cultures with inhibitors, whereas differences between tumor cells alone and co-cultures with/without inhibitors were statistically significant (P < 0.05, ANOVA and Bonferroni’s post test).