Figure 4. Diminished vascular density of B16 melanoma tumors in CL1-R2–treated mice. (A) Immunohistochemical assessment of vascular density in B16 tumor sections taken on day 16 from mice treated with control IgG1 (Ctrl) or CL1-R2, both in combination with cyclophosphamide (CycloP). Endothelial cells in representative sections were stained with isolectin B4 (brown) and counterstained with hematoxylin (blue). Images are representative of five mice in each group. Bar, 50 µm. (B) Quantification of the data shown in A. Vessel density was counted in four independent areas on each B16 tumor section. ***, P < 0.0001 (one-way ANOVA test). Error bars represent SEM. (C) 3 d after B16 cell injection, mice received repeated i.p. injections of CL1-R2 or IgG1 (Ctrl). Cyclophosphamide was then added to the drinking water. Mice were examined by DUS to identify intratumoral blood vessels 12, 15, and 18 d after the first CL1-R2 injection. Power Doppler mode color DUS ultrasound images with intratumoral blood vessel signals are shown in red/yellow. Representative images obtained from the same tumor cross section in a single mouse from day 12 to 18 are shown for each treatment. Bar, 5 mm. Side bar: Doppler power, maximum 4.3. (D) Quantification of the number of intratumoral vessels in IgG1-treated (black bars) and CL1-R2–treated (gray bars) mice determined from multiple cross sections of the tumor. Tumor vessel number on the ordinate represents the mean number of total intratumoral blood vessels per tumor because the whole tumor was scanned at each time point for the detection of blood vessels. Data are expressed as means ± SEM (n = 5 mice in each group). *, P = 0.0203 for day 12; **, P = 0.0032 for day 15; **, P = 0.0077 for day 18 (Student’s t test). (E) B16 tumor growth in untreated (PBS), CL1-R2–treated (500 µg, three times a week), or control mice in combination with cyclophosphamide treatment. Tumor volumes were measured at the indicated times after tumor inoculation. Data from four experiments (n = 5 mice/group) were pooled. Data represent mean tumor size values ± SEM. ***, P < 0.0001 (two-way ANOVA test).
Figure 4.

Diminished vascular density of B16 melanoma tumors in CL1-R2–treated mice. (A) Immunohistochemical assessment of vascular density in B16 tumor sections taken on day 16 from mice treated with control IgG1 (Ctrl) or CL1-R2, both in combination with cyclophosphamide (CycloP). Endothelial cells in representative sections were stained with isolectin B4 (brown) and counterstained with hematoxylin (blue). Images are representative of five mice in each group. Bar, 50 µm. (B) Quantification of the data shown in A. Vessel density was counted in four independent areas on each B16 tumor section. ***, P < 0.0001 (one-way ANOVA test). Error bars represent SEM. (C) 3 d after B16 cell injection, mice received repeated i.p. injections of CL1-R2 or IgG1 (Ctrl). Cyclophosphamide was then added to the drinking water. Mice were examined by DUS to identify intratumoral blood vessels 12, 15, and 18 d after the first CL1-R2 injection. Power Doppler mode color DUS ultrasound images with intratumoral blood vessel signals are shown in red/yellow. Representative images obtained from the same tumor cross section in a single mouse from day 12 to 18 are shown for each treatment. Bar, 5 mm. Side bar: Doppler power, maximum 4.3. (D) Quantification of the number of intratumoral vessels in IgG1-treated (black bars) and CL1-R2–treated (gray bars) mice determined from multiple cross sections of the tumor. Tumor vessel number on the ordinate represents the mean number of total intratumoral blood vessels per tumor because the whole tumor was scanned at each time point for the detection of blood vessels. Data are expressed as means ± SEM (n = 5 mice in each group). *, P = 0.0203 for day 12; **, P = 0.0032 for day 15; **, P = 0.0077 for day 18 (Student’s t test). (E) B16 tumor growth in untreated (PBS), CL1-R2–treated (500 µg, three times a week), or control mice in combination with cyclophosphamide treatment. Tumor volumes were measured at the indicated times after tumor inoculation. Data from four experiments (n = 5 mice/group) were pooled. Data represent mean tumor size values ± SEM. ***, P < 0.0001 (two-way ANOVA test).

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