FIGURE 4.

PEDF causes a TRAIL-mediated antitumor effect in a murine model. A, PEDF induces TRAIL expression in stromal macrophages. C57BL/6 mice with established TC-1 tumors were intraperitoneally injected with GW9662 or DMSO vehicle for 6 h, followed by peritumoral injections with PEDF (P) or PEDF solvent (S) control for a further 24 h as described under “Experimental Procedures.” After treatment, the mice were euthanized, and the tumors (n = 6) were processed for macrophage isolation. The freshly isolated macrophages were then double-stained with PE-conjugated anti-TRAIL and PerCP-conjugated anti-F4/80 antibodies for flow cytometry analysis. *, p < 0.02 versus solvent; #, p < 0.001 versus DMSO + PEDF. B, TRAIL-R2-Fc blocks PEDF-induced TC-1 cell apoptosis. C57BL/6 mice bearing TC-1 tumors were pretreated with or without GW9662 for 6 h and then injected around the tumor with PEDF or PEDF combined with TRAIL-R2-Fc or control IgG1. At day 2 post-treatment, tumors were harvested, and tumor sections were double-stained with TUNEL to identify apoptotic cells (green) and F4/80 to identify macrophages (red). Representative photographs revealed PEDF-induced tumor cell apoptosis in the peripheral tumor and in the vicinity of macrophages. Apoptotic TC-1 cells were quantified under a microscope (×400, 10 fields/tumor section) using a digital program. Data are representative of three individual experiments. *, p < 0.001 versus solvent. **, p < 0.001 versus PEDF. #, p < 0.005 versus PEDF + IgG1. Error bars, S.E.