Fig. 2
CAFs induce the polarization of THP-1 cells into anti-inflammatory M2-like macrophages. A After 24 h of incubation with 150 nM PMA, the monocyte-to-macrophage differentiation was confirmed by the elevated transcript levels of recognized macrophage markers CD36, CD68, and CD71. The decreased transcript level of CD14 in PMA-treated THP-1 cells further confirmed that the THP-1 monocytes were differentiated into macrophage-like cells. B Transcript expression levels of M2 macrophage markers CD163 and CD206 in THP-1 macrophages incubated with CAF-CM were significantly increased compared with those in THP-1 M0 macrophages alone or incubated with NF-CM after 48 h. As a positive control, THP-1 macrophages were stimulated with 20 ng/mL of IL-4 for 48 h. C Protein secretion levels of the anti-inflammatory cytokine IL-10 and the pro-inflammatory cytokine IL-12 were assessed by ELISA. THP-1 macrophages incubated with CAF-CM as well as positive control M2 macrophages were identified by IL-10high IL-12low phenotype when compared to THP-1 M0 macrophages alone or incubated with NF-CM, confirming the potential of CAFs in promoting the polarization of THP-1 cells into anti-inflammatory M2-like macrophages after 48 h. D, E The relative levels of intracellular ROS production during macrophage polarization. The amount of ROS produced by differentiated THP-1 macrophages increased noticeably when monocytes were treated with PMA. Importantly, ROS generation in CAF-educated macrophages was lower than that in macrophages educated by NF-CM after 48 h. Even though the level of ROS diminishes during M1/M2 polarization, CAF-induced M2-like macrophages were found to produce higher levels of ROS than THP-1 control monocytes. Representative flow cytometry histograms (D) and bar graphs of relative DCFDA fluorescence intensity (E) imply that CAFs contribute to M2 polarization in part by increasing ROS production, as M2 macrophages require a level of ROS for proper polarization. Columns, mean of three different experiments; bars, SD. ns: non-significant, *P-value < 0.05, **P-value < 0.01, ***P-value < 0.001