Fig. 3.

PLMDs induced M1 macrophage polarization in normoxic and hypoxic RAW264.7 macrophages in vitro and reduced the expression of PD-L1 in PC3 and TRAMP-C2 tumors in vivo. Immunofluorescence images of (A) CD86 (M1 macrophage marker), and (B) M2 macrophages marker, CD163, in RAW264.7 cells. Scale bars, 50 μm. (C) TNF-α release in the supernatant of RAW264.7 cells treated with 12.5 μM PLMDs for 24 h under normoxic or hypoxic conditions. (D) Illustration of Transwell assay for coculturing study as explained in the materials and methods. (E) Microscope images of viable TRAMP-C2 cells stained with crystal violet in the presence and absence of RAW264.7 cells or PLMDs. Scale bars, 100 μm. (F and G) Survival of TRAMP-C2 cells cocultured with or without RAW264.7 cells and/or PLMD treatment. (H) IHC images for the ex-vivo PD-L1 expression 5 d after saline, PLMDs (0.87 mg MnO₂/kg bw), RT alone (10 Gy), and PLMDs + RT treatment in PC3 and TRAMP-C2 tumors. Scale bars, 200 μm. (I and J) Corresponding quantification of the percentages of PD-L1-expressing cells after various in vivo treatments. Saline n = 3, PLMDs alone n = 4, RT alone n = 4, PLMDs + RT n = 5. Data are expressed as means ± SD. P < 0.05 (*), P < 0.005 (**), P < 0.0005 (***), and P < 0.0001 (****).