Endocrine vasculatures are preferable targets of an antitumor ineffective low dose of anti-VEGF therapy

Abstract

Anti-VEGF-based antiangiogenic drugs are designed to block tumor angiogenesis for treatment of cancer patients. However, anti-VEGF drugs produce off-tumor target effects on multiple tissues and organs and cause broad adverse effects. Here, we show that vasculatures in endocrine organs were more sensitive to anti-VEGF treatment than tumor vasculatures. In thyroid, adrenal glands, and pancreatic islets, systemic treatment with low doses of an anti-VEGF neutralizing antibody caused marked vascular regression, whereas tumor vessels remained unaffected. Additionally, a low dose of VEGF blockade significantly inhibited the formation of thyroid vascular fenestrae, leaving tumor vascular structures unchanged. Along with vascular structural changes, the low dose of VEGF blockade inhibited vascular perfusion and permeability in thyroid, but not in tumors. Prolonged treatment with the low-dose VEGF blockade caused hypertension and significantly decreased circulating levels of thyroid hormone free-T3 and -T4, leading to functional impairment of thyroid. These findings show that the fenestrated microvasculatures in endocrine organs are more sensitive than tumor vasculatures in response to systemic anti-VEGF drugs. Thus, our data support the notion that clinically nonbeneficial treatments with anti-VEGF drugs could potentially cause adverse effects.

Keywords: VEGF; adverse effects; angiogenesis; antiangiogenic therapy; tumor.

Fig. 1.

Dose-dependent effects of VEGF blockade on tumor growth and angiogenesis. (A, C, and E) Dose-dependent effects of VEGF blockade on T241 (A), LLC (C), and HT29 (E) tumor growth after a 2-wk systemic treatment schedule. Doses of 2.5 mg/kg and 5.0 mg/kg produced significantly inhibited tumor growth in all three tumor models. VEGF blockade at doses of 0.5 mg/kg and 1.5 mg/kg did not significantly inhibited tumor growth. (B, D, and F) CD31⁺ microvessel density in VEGF blockade- or nonimmune IgG-treated T241 (B), LLC (D), and HT29 (F) tumors and quantified from eight random fields per group. Six to eight mice per group were used. NS, not significant; *P < 0.05; **P < 0.01. (Scale bars, 50 μm.) Quantitative data are presented as mean determinants ± SEM.

Fig. 2.

Impact of VEGF blockade on thyroid vasculatures in tumor-bearing mice. T241 (A), LLC (B), and HT29 (C) tumor-bearing mice were systemically treated with nonimmune IgG and various dosages of VEGF blockades and stained with CD31 and H&E. CD31⁺ microvessels were quantified from eight random fields per group. Six to eight mice per group were used. NS, not significant; **P < 0.01; ***P < 0.001. (Scale bars, 50 μm.) Quantitative data are presented as mean determinants ± SEM.

Fig. S1.

Impact of VEGF blockade on adrenal gland vasculatures in tumor-bearing mice. T241 (A), LLC (B), and HT29 (C) tumor-bearing mice were systemically treated with nonimmune IgG and various dosages of VEGF blockades and stained with CD31 and H&E. CD31⁺ microvessels were quantified from eight random fields per group. Six to eight mice per group were used. NS, not significant; *P < 0.05; ***P < 0.001. (Scale bars, 50 μm.) Quantitative data are presented as mean determinants ± SEM.

Fig. S2.

Impact of VEGF blockade on pancreatic β-islet vasculatures in tumor-bearing mice. T241 (A), LLC (B), and HT29 (C) tumor-bearing mice were systemically treated with nonimmune IgG and various dosages of VEGF blockades and stained with CD31 and H&E. CD31⁺ microvessels were quantified from eight random fields per group. Six to eight mice per group were used. NS, not significant; *P < 0.05; **P < 0.01; ***P < 0.001. (Scale bar, 50 μm.) Quantitative data are presented as mean determinants ± SEM.

Fig. 3.

VEGF blockade-induced alterations of vascular fenestrations, permeability, perfusion, and hypoxia in thyroid glands of T241 tumor-bearing mice. (A) Inhibition of thyroid, but not tumor, endothelium fenestrations by all doses of VEGF blockades. Nonimmune IgG was used as a control. Fenestrae numbers were quantified as per micrometer from 8 to 10 vessels per group. Arrows point to endothelium fenestrae. (Scale bar, 250 nm.) (B) Leakage of fluorescein-labeled 70-kDa dextran (green) in tumors and thyroid gland. Thyroid vasculatures were stained with CD31 (red). Arrowheads point to extravasated fluorescein-dextran molecules. (Scale bar, 50 μm.) Data were quantified from eight random fields per group (n = 6–8 mice per group). (C) Perfusion of fluorescein-labeled 2,000-kDa dextran (green) in tumors and thyroid gland. Thyroid vasculatures were stained with CD31 (red). (Scale bar, 50 μm.) Data were quantified from eight random fields per group (n = 6–8 mice per group). (D) Tumor and thyroid tissue hypoxia was measured by pimonidazole positive signals. (Scale bar, 50 μm.) Data were quantified from eight random fields per group (n = 6–8 mice per group). NS, not significant; *P < 0.05; **P < 0.01; ***P < 0.001. Quantitative data are presented as mean determinants ± SEM.

Fig. 4.

Prolonged anti-VEGF treatment at 1.5 mg/kg reduces thyroid free T4 hormone. (A) Anti-VEGF 4-wk treatment at 1.5 mg/kg did not inhibit EO771 tumor growth compared with nonimmune IgG (n = 6–8 mice per group). (B) Microvascular changes in tumors, thyroid glands, adrenal glands, and pancreatic β-islets. (Scale bar, 50 μm.) Data were quantified from eight random fields per group (n = 6–8 mice/group). (C and D) Measurement of thyroid free-T3 and T4 hormones, cortisol, and insulin in plasma of nonimmune IgG- and anti-VEGF–treated mice (n = 6–8 samples per group). NS, not significant; *P < 0.05; **P < 0.01; ***P < 0.001. Quantitative data are presented as mean determinants ± SEM.

Fig. 5.

Prolonged anti-VEGF therapy causes functional changes in endocrine organs in a tumor-free mouse model. (A) Microvascular changes in thyroid glands, adrenal glands, and pancreatic β-islets in tumor-free mice after treatment with VEGF blockade for 12-wk at 1.5 mg/kg compared with nonimmune IgG (n = 6–8 mice per group). (Scale bar, 50 μm.) Data were quantified from eight random fields per group (n = 6–8 mice per group). (B) Systolic and diastolic blood pressure changes after 12-wk 1.5 mg/kg VEGF blockade treatment (n = 6–8 mice per group). However, 1.5 mg/kg VEGF blockade has no impact on blood pressures after 2-wk short-term treatment (n = 6–8 mice per group). (C) Measurements of plasma free-T3 and -T4 thyroid hormones after 12-wk anti-VEGF treatment (n = 6–8 mice per group). (D) Measurements of plasma glucocorticoid hormones after 12-wk anti-VEGF treatment (n = 6–8 mice per group). (E) Measurements of plasma insulin levels after 12-wk anti-VEGF treatment (n = 6–8 mice per group). NS, not significant; *P < 0.05; **P < 0.01; ***P < 0.001. Quantitative data are presented as mean determinants ± SEM.