Figure 1.
Regression of tumor vasculature after endostar treatment.
CNE-2 and 5–8F tumors were removed and stained with anti-CD31 antibody. Tumor blood vessels were analyzed by confocal microscopy at different days after treatment as indicated. Tumor blood vessels are presented in red. A-a: CD31 had an irregular distribution in control tumors after 9 days of treatment with vehicle in CNE-2 tumor. A-b: After the treatment with endostar for 9 days, reduction in CD31 in CNE-2 tumor was reflected by decreased immunofluorescence. B–C: Quantification of CD31-positive tumor vessels. Bar graphs illustrated changes in area density of CD31-positive vessels in CNE-2 and 5–8F tumors. Quantificatoin were determined from 9–12 randomized cryosectioned fields (n = 4–6 mice per group). Columns, means; bars, SEM. *p<0.05 compared with the control group, * *p<0.01 compared with control (Student's t-tests). Scale bar: 50 µm.
Figure 2.
Confocal microscopic images showing comparison of vascular basement membrane in NPC tumors with or without endostar treatment.
Fluorescence images of tumors showed CD31-positive endothelial cells (red), type IV collagen-positive basement membrane (green), and merged images (orange). A: Some segments of CD31 lacked type IV collagen immunoreactivity in untreated CNE-2 tumors (a–c). Most CD31 coincided with regions of type IV collagen-positive blood vessels 5 days after endostar treatment in CNE-2 tumors (d–f). B–C: Graphs of the percentage of type IV collagen showed that basement membrane was increased during regression of endothelial cells in both CNE-2 and 5–8F tumors. Quantification of type IV collagen-positive vessels versus total CD31-positive vessels were determined from 9–12 randomized cryosectioned fields (n = 4–6 mice per group). Columns, means; bars, SEM. *p<0.05 compared with the control group, * *p<0.01 compared with control (Student's t-tests). Scale bar: 50 µm.
Figure 3.
Confocal microscopic images showing comparison of pericyte coverage in NPC tumors with or without endostar treatment.
Fluorescence images of tumors showed CD31-positive endothelial cells (red), NG2-positive pericytes (green), and merged images (orange). A: Some segments of CD31 lacked NG2 immunoreactivity in untreated CNE-2 tumors (a–c). The intensity of NG2 immunofluorescence colocalized with CD31 staining were increased 5 days after endostar treatment in CNE-2 tumors (d–f). B–C: Graphs of the percentage of NG2 showed that pericytes were increased during regression of endothelial cells in both CNE-2 and 5–8F tumors. Quantification of percentages of NG2-positive vessels versus total CD31-positive vessels was determined from 9–12 randomized cryosectioned fields (n = 4–6 mice per group). Columns, means; bars, SEM. *p<0.05 compared with the control group, * *p<0.01 compared with control (Student's t-tests). Scale bar: 50 µm.
Figure 4.
Confocal microscopic images showing comparison of tumor hypoxia in NPC tumors with or without endostar treatment.
Tumor hypoxia (pimonidazole staining, green) was severe in control tumors (A-a), but decreased on day 5 during monotherapy with endostar (A-b) in CNE-2 tumor. Hypoxia reached a minimum at day 5, and a partial relapse occurred at day 9 in CNE-2 tumor (B), while hypoxia reached a minimum at day 3, and a relapse occurred at day 7 in 5–8F tumor (C). Quantification of hypoxic tumor fraction was determined from the whole tumor cryosectioned fields (n = 4–6 mice per group). Columns, means; bars, SEM. *p<0.05 compared with the control group, * *p<0.01 compared with control (Student's t-tests). Scale bar: 1 mm.
Figure 5.
Effect of the combination of endostar and radiation on NPC tumor growth.
Subcutaneously implanted CNE-2 and 5–8F xenografted tumors were established as described in Materials and Methods. When xenografted tumors reached a volume of 100 mm3, mice in CNE-2 xenografts were randomly divided into six groups (n = 6–8 per group): con, control (0.9% saline); endo, endostar only; R, radiation at a single dose of 6 Gy only; endo+R3, radiation at a single dose of 6 Gy on the 3rd day after endostar treatment; endo+R5, radiation at a single dose of 6 Gy on the 5th day after endostar treatment; endo+R9, radiation at a single dose of 6 Gy on the 9th day after endostar treatment. Mice in 5–8F xenografts were randomly divided into five groups (n = 6–8 per group): con, control; endo, endostar only; R, radiation at a single dose of 6 Gy only; endo+R3, radiation at a single dose of 6 Gy on the 3rd day after endostar treatment; endo+R7, radiation at a single dose of 6 Gy on the 7th day after endostar treatment. Endostar was administered at a dose of 20 mg/kg/d for 10 days from the first day of treatment. A, C: The growth curve of CNE-2 and 5–8F tumor xenografts. Radiation induced a significantly tumor suppression when combining with endostar on day 5 and day 3. B, D: A combination of radiation and endostar is synergistic only during a “normalization window” when tumor hypoxia is greatly diminished. Each point represents the mean tumor size. Columns, means; bars, SEM. *p<0.05 compared with the control group, * *p<0.01 compared with control (ANOVA, LSD-t test).
Figure 6.
Immunohistochemical staining and quantitative analysis for VEGF, PEDF, MMP-2, MMP-9, and MMP-14 in human CNE-2 xenograft tissues.
Xenografts were harvested at day 5 after treatment initiation. The levels of VEGF, MMP-2, MMP-9 and MMP-14 were decreased, while the level of PEDF was increased in CNE-2 xenografts treated with endostar for 5 days compared with the control group (Student's t-tests). a–c, VEGF staining and quantitative data; d–f, PEDF immunostaining and quantitative data; g–i, MMP-2 immunostaining and quantitative data; j–l, MMP-9 immunostaining and quantitative data; m–o, MMP-14 immunostaining and quantitative data. n = 6/group. *p<0.05 compared with control, * *p<0.01 compared with control (Student's t-tests). Scale bar: 100 µm.