Inhibition of osteosarcoma cell growth by LPS + IFN-γ–activated M1-like macrophages. Human M1-like macrophages (dark shade) and M2-like macrophages (light shade) were pre-activated with or without LPS + IFN-γ (M1 and M2) or IL-10 (M2) and afterwards incubated with (A) HOS-143b cells (n = 4–11) and (B) OHS cells (n = 5–12) and (C) four other osteosarcoma cell lines (n = 2–12). After two days of co-culture, tumor cell numbers were counted by flow cytometry. Differences between one macrophage–tumor co-culture and the control, i.e. tumor cell recovery after incubation in the absence of macrophages (white bar, set to 100%), as in panel C were statistically analyzed by paired student t-tests, *** is P < 0.001, ** is P < 0.01, * is P < 0.05, ns is not statistically significant. Differences between multiple groups as in panel A and B were statistically analyzed by ANOVA as indicated followed by Dunnett’s post test for differences (p < 0.05) between individual co-cultures and the control as indicated by asterisks. (D) HOS-143b cell counts after 2, 24 and 48 hours co-culture with M1-like macrophages +/− LPS + IFN-γ (n = 5). All data are means of multiple experiments as indicated (n).