Host-derived angiopoietin-2 affects early stages of tumor development and vessel maturation but is dispensable for later stages of tumor growth

Abstract

The angiopoietin/Tie2 system has been identified as the second vascular-specific receptor tyrosine kinase system controlling vessel assembly, maturation, and quiescence. Angiopoietin-2 (Ang-2) is prominently up-regulated in the host-derived vasculature of most tumors, making it an attractive candidate for antiangiogenic intervention. Yet, the net outcome of Ang-2 functions on tumor angiogenesis is believed to be contextual depending on the local cytokine milieu. Correspondingly, Ang-2 manipulatory therapies have been shown to exert protumorigenic as well as antitumorigenic effects. To clarify the role of Ang-2 for angiogenesis and tumor growth in a definite genetic experimental setting, the present study was aimed at comparatively studying the growth of different tumors in wild-type and Ang-2-deficient mice. Lewis lung carcinomas, MT-ret melanomas, and B16F10 melanomas all grew slower in Ang-2-deficient mice. Yet, tumor growth in wild-type and Ang-2-deficient mice dissociated during early stages of tumor development, whereas tumor growth rates during later stages of primary tumor progression were similar. Analysis of the intratumoral vascular architecture revealed no major differences in microvessel density and perfusion characteristics. However, diameters of intratumoral microvessels were smaller in tumors grown in Ang-2-deficient mice, and the vasculature had an altered pattern of pericyte recruitment and maturation. Ang-2-deficient tumor vessels had higher pericyte coverage indices. Recruited pericytes were desmin and NG2 positive and predominately alpha-smooth muscle actin negative, indicative of a more mature pericyte phenotype. Collectively, the experiments define the role of Ang-2 during tumor angiogenesis and establish a better rationale for combination therapies involving Ang-2 manipulatory therapies.

Figure 1

Growth of LLC in WT and Ang-2–deficient mice. LLC tumors grew significantly slower in Ang-2–deficient mice (A). Tumor growth dissociated during early tumor growth when the tumors had grown to 0.2 to 0.4 cm³. Thereafter, tumor growth rates were almost identical as evidenced by essentially parallel curves of log-transformed tumor growth data (B). Total tumor weight was reduced in tumors grown in Ang-2–deficient mice (C). Yet, the differences in tumor weight at the end of the experiment resulted mostly from the dissociating exponential growth curves as a consequence of a dissociation of tumor growth curves during early stages of tumor growth. Tumors grown in WT mice grew cuboidal with almost spherical three-dimensional characteristics. In contrast, tumors grown in Ang-2–deficient mice had a flattened appearance (D). *, P < 0.001, compared with WT mice.

Figure 2

Effect of Ang-2 deficiency on mural cell recruitment and maturation during early stages of LLC growth. LLC tumor cells were s.c. injected in WT and Ang-2–deficient mice. Tumors were harvested 7 d (A) and 12 d (B) after tumor inoculation. Tumor sections were double-stained for the endothelial cell marker CD31 (green) and for the mural cell markers NG2, desmin, and α-SMA (red). Vessel coverage was calculated as the percentage of NG2-, desmin-, and α-SMA–positive vessels compared with the number of CD31-positive vessels. Mural cell coverage was similar in tumors grown in WT and Ang-2–deficient mice at day 7 (A). In contrast, day 12 tumors grown in Ang-2–deficient mice had higher coverage by NG2- and desmin-positive and less coverage by α-SMA–positive mural cells (B). n.d., not detected. *, P < 0.05; **, P < 0.01. Bar, 100 μm.

Figure 3

Effect of Ang-2 deficiency on mural cell recruitment and maturation during later stages of LLC growth. Tumors were grown s.c. in WT and Ang-2–deficient mice and harvested when the first tumors had grown to 2 cm³. Tumor sections were double-stained for the endothelial cell marker CD31 (green) and for the mural cell markers NG2, desmin, and α-SMA (red). Vessel coverage was calculated as the percentage of NG2-, desmin-, and α-SMA–positive vessels compared with the number of CD31-positive vessels. As in day 12 mice, late-stage LLC tumors grown in Ang-2–deficient mice had higher coverage by NG2- and desmin-positive and less coverage by α-SMA–positive mural cells. *, P < 0.05; **, P < 0.01. Bar, 100 μm.

Figure 4

Effect of host-derived Ang-2 deficiency on MVD (A and B), diameter of intratumoral microvessels (A and B), and perfusion (C) in LLC tumors. Tumors were grown in WT and Ang-2–deficient mice and harvested when the first tumors had grown to 2 cm³. MVDs and vessel diameters were quantitated in CD31-stained tissue sections. Perfusion was assessed on the basis of FITC-lectin perfusion labeling. Ang-2 deficiency had a nonsignificant effect on intratumoral MVD. Yet, the average diameters of intratumoral microvessels were significantly reduced in tumors grown in Ang-2–deficient mice. Correspondingly, intratumoral microvessels in Ang-2–deficient mice were better perfused. **, P < 0.01. Bar, 200 μm.

Figure 5

Growth of MT-ret melanomas in WT and Ang-2–deficient mice. Tumor growth in MT-ret melanomas dissociated during the very earliest stages of tumor growth (A). Thereafter, tumor growth rates were almost identical as evidenced by essentially parallel curves of log-transformed tumor growth data (B). Total tumor weight was strongly reduced in tumors grown in Ang-2–deficient mice (C). Yet, as in LLC tumors, the differences in tumor weight at the end of the experiment resulted mostly from the dissociating exponential growth curves as a consequence of a dissociation of tumor growth curves during early stages of tumor growth. *, P < 0.05.

Figure 6

Effect of Ang-2 deficiency on MVD, vessel diameter and perfusion (A), and mural cell recruitment and maturation (B) in MT-ret melanomas. MT-ret melanoma cells were s.c. injected in WT and Ang-2–deficient mice and harvested when the first tumors had grown to 2 cm³. MVDs and vessel diameters were quantitated in CD31-stained tissue sections. Perfusion was assessed on the basis of FITC-lectin perfusion labeling. For mural cell coverage analysis, tumor sections were double-stained for the endothelial cell marker CD31 and for the mural cell markers desmin (top), NG2 (middle), and α-SMA (bottom). Vessel coverage was calculated as the percentage of desmin-, NG2-, and α-SMA–positive vessels compared with the number of CD31-positive vessels. As in LLC tumors, microvessels in MT-ret melanomas had higher desmin and NG2 coverage and lower αSMA coverage in tumors grown in Ang-2–deficient mice.