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. Author manuscript; available in PMC: 2017 Jul 11.
Published in final edited form as: Oncogene. 2017 Feb 20;36(26):3749–3759. doi: 10.1038/onc.2017.1

Figure 3. In cultured cells, MIF promotes M1 polarization, causing macrophages to be less phagocytic and exert a less pro-tumoral effect.

Figure 3

(a) Murine bone marrow-derived macrophage precursors were matured for 5 days and rested for 3 days prior to an 18 hour polarization in media containing 200 ng/mL and 800 ng/mL MIF, 20 ng/mL IFN-γ (for M1 polarization), 20 ng/mL IL-4 (for M2 polarization), or conditioned media from U87-BevR or U87-BevS cells (n=3/group). M1/M2 polarization ratio was assessed by multiplication of the qPCR fold increases in three different M1 primers divided by the qPCR fold increases in three different M2 primers normalized to results when incubating with media alone. Recombinant MIF drove M1 polarization in a dose-dependent manner (p<0.0001), while, unlike media from U87-BevS cells, media from U87-BevR cells drove an M2 macrophage polarization (P<0.0001). (b) The phagocytic activity of THP-1 monocyte-derived macrophages without stimulation, cultured with cytokines (recombinant MIF, IFN-γ, and IL-4), or cultured with conditioned media (CM) was assessed via uptake of fluorescent heat-killed E. coli with subsequent measurement of the proportion of fluorescent cells (n=6/group). M1 polarized macrophages were less phagocytic whereas M2 polarized macrophages were more phagocytic relative to unstimulated control macrophages. Macrophages treated with 800 ng/mL MIF were less phagocytic relative to control (P=0.005). Macrophages treated with CM from U87-BevR cells were more phagocytic than macrophages treated with CM from U87-BevS (P=0.01) (example images shown to the right). (c) Sequential conditioned media (SCM) experiments were performed as illustrated to the left. Briefly, media from U87-BevR or U87-BevS cells was applied to THP-1-derived macrophages, and the media was then taken from those macrophages and applied to U87 cells, with numbers of U87 cells counted 48 hours later (n=24/group). U87-BevR-derived macrophage conditioned media (U87-BevR MCM) stimulated a significantly greater expansion of U87 cells than U87-BevS-derived macrophage conditioned media (U87-BevS MCM) (P<0.0001), with U87-BevR MCM and U87-BevS MCM stimulating more (P=0.005) and less (P=0.003) U87 expansion than control media, respectively.