D. Quantitative

results for microvessel density (MVD) in tumor tissue. Ad-PEDF CFTR modulator group shows a significant decrease of MVD compared to control groups (p < 0.05). E. Micrographs show tumor tissue sections stained with H&E. Decreased density of vessels and noticeable necrosis was observed in tumors from Ad-PEDF treated mice (c). In contrast, tumor cells grew well with less necrosis in NS (a) or Ad-Null group (b). (Original magnification, ×400). n = 2; 3 sections/mouse. To further determine whether the increase in apoptosis of Ad-PEDF treated tumor tissue was associated with the antiangiogenic effect of PEDF, we analyzed MVD of tumor tissues in each group. As shown in Fig 5C, intensive CD31 immunoreactive microvessels was observed in tumor tissue from mice treated by NS and Ad-null, but only moderate CD31 staining present in tumor tissue from mice treated by Ad-PEDF. For comparison, CD31-positive single or a cluster of cells were counted as the microvessels, and MVD was calculated for each group with the formula described in the materials and methods. MVD of tumor tissues from Ad-PEDF treated mice

exhibited a significant decrease than from Ad-null or NS treated mice, (21 ± 4, 54.3 ± 7.2, 62 ± 6.5, respectively) (p < 0.05, Fig 5D). These data suggest that the decreased angiogenesis after MG-132 manufacturer Ad-PEDF treatment may be responsible for the increased apoptosis. Adjacent O-methylated flavonoid sections were stained with H&E to evaluate the morphologic changes after Ad-PEDF or control treatments. Consistent with the results of CD31 immunochemistry staining, less vessels and remarkable necrosis areas were observed in tumor tissue from Ad-PEDF treated mice in comparison to Ad-null or NS treated mice (Fig 5E). Collectively, these data suggest that serum PEDF from infected host cells is sufficient to inhibit

tumor angiogenesis, subsequently promote apoptosis, reduce tumor progression and prolong survival time. Ad-PEDF treatment inhibited the development of tumor angiogenesis To confirm the proceeding finding that PEDF from Ad-PEDF gene transfer is associated with the reduction of tumor angiogenesis, and to directly demonstrate the causal relationship, we performed the alginate-encapsulated tumor cell assay, which is capable of demonstrating whether the development of tumor angiogenesis is prevented by PEDF treatment. As shown in Fig 6, the intensity of blood vessels on the surface of tumor cell-containing alginate beads was noticeably less in Ad-PEDF-treated mice than Ad-null or NS treated mice (Fig 6A). The quantification results for the amount of FITC-dextran indicate that the distribution of extravasated tracer in the encapsulated tumor tissues was consistent with the distribution of blood vessels on the bead surface; the amount of FITC-dextran per beads in Ad-PEDF, Ad-null and NS group was 2.1 ± 0.3 μg/bead, 5.8 ± 0.3 μg/bead and 6.2 ± 0.6 μg/bead, respectively.