Angiopoietin-2-blocking Antibody and Lung Metastasis
Angiopoietin-2-blocking Antibody and Lung Metastasis
Our results indicate that Ang2 increases tumor metastasis at least in part by promoting endothelial disruption and increasing tumor cell translocation and homing to target organs. We also provide mechanistic insight into the effects of the antibody-mediated blockade of Ang2 at the cellular level. To address the function of Ang2 in tumor metastasis, we used systemic and endothelial cell-specific Ang2 overexpression, as well as Ang2-blocking antibodies in various tumor models. Our results demonstrate that systemic Ang2 overexpression promotes tumor lymphangiogenesis as well as lymph node and lung metastasis in addition to tumor growth and angiogenesis. Ang2 also enhanced the establishment of metastatic colonies in the lungs after intravenous tumor cell inoculation. Importantly, by using VEC-tTA/Tet-OS-Ang2 transgenic mice, in which Ang2 is specifically induced only in the vascular endothelium, we demonstrated that endothelial overexpression of Ang2 increases lung metastasis. Alternatively, Ang2 blockade attenuated tumor lymphangiogenesis, dissemination of tumor cells via the lymphatic vessels, lung metastasis, and tumor cell colonization of the lungs.
The antiangiogenic effects of specific Ang2 blockade on tumor growth and vascularization have been previously characterized using Ang2 blocking in various tumor xenografts, but the effects on tumor metastasis were not investigated. In one report, intrapleural injection of lentiviral Ang2 short hairpin RNA in mice bearing mammary tumor xenografts was reported to inhibition endothelial permeability in the lungs. In another report, Ang2 overexpression in tumor cells increased their invasive properties by an autocrine, Tie2-independent effect mediated via the integrins. However, only a minor fraction of human tumor cell lines express detectable amounts of Ang2. Importantly, the lung cancer cells we used expressed no detectable Ang2 in culture, unlike in previous studies in which Ang2 inhibitors were used.
Targeted deletion of Ang2 leads to defective blood vessel development in the eye and lymphatic vessel development in the mesenterium and skin. In the developing postnatal retina, the Ang2-blocking antibody was found to delay vascularization, which was confirmed in our study. Further analysis showed that blocking Ang2 induced vascular tuft-like structures at the angiogenic front, where signs of sporadic sprout regression were observed. In the primary tumors treated with the Ang2-blocking antibody, basement membrane and pericyte marker–positive and endothelial cell negative vessel structures were present, similarly indicating endothelial regression. These results are consistent with the data that autocrine Ang2 contributes to endothelial cell survival and migration in some contexts (10,42, our unpublished results using Ang2-blocking antibody). In mice surviving Ang2 deletion, an initial delay was observed in the growth of isogenic tumors; however, in our study, the Ang2-blocking antibodies also induced statistically significant inhibition in the later phases of tumor growth. Thus, it is possible that genetic deletion of Ang2 and blocking antibodies against Ang2 have different mechanisms of action.
We and Fukuhara et al. have previously shown that angiopoietins induce the formation of unique Tie2 receptor complexes in endothelial cells. In cells with established contacts to other cells, Ang1 and Ang2 were found to trigger Tie2 translocation to cell–cell junctions, where Ang1 has been shown to reduce paracellular permeability. Here, we show that autocrine Ang2 secreted by endothelial cells similarly induces Tie2 and Tie1 translocation to cell–cell contacts. However, in contrast to Ang1, Ang2 induced only weak Tie2 phosphorylation and may thus decrease Ang1 signals via the Tie receptors in the endothelial cell–cell junctions. When the endothelial cells were cocultured with the tumor cells, the presence of Ang2-Tie2 complexes was increased in the endothelial cell–cell junctions, even when the cells were under normal oxygen pressure. The Ang2-blocking antibody inhibited endothelial Ang2 function by inducing the internalization of the Ang2-Tie2 receptor complexes and by inhibiting the subsequent formation of such complexes. Because of the multimeric nature of Ang2, the anti-Ang2 antibody may result in the formation of large receptor clusters on the cell surface; these could be more effectively targeted for endocytosis. The Ang1-induced Tie receptor complexes were not affected by the Ang2 antibody. Because only the Ang2-bound Tie2 receptor fraction was directed for internalization, the results suggest that during Ang2 blocking in the metastatic lungs, Ang1 can bind and activate Tie2, contributing to stabilization of endothelial cell–cell junctions.
Our results showed that fewer tumor cell colonies were established in the lungs of mice treated with the Ang2-blocking antibody when compared with control antibody–treated mice at the relatively early time points when angiogenesis was not yet detected in the metastatic foci. Although we have not imaged extravasation per se, these results suggest that blocking Ang2 inhibited the extravasation of tumor cells to the lungs or the very early stages of metastatic colony establishment. It was previously shown that the tumor cell–associated lung capillaries express increased amounts of Ang2. In the pulmonary vessels associated with the lung metastases, the Ang2-blocking antibody seemed to counteract the disruption of the endothelial cell–cell junctions and vascular integrity, suggesting that by decreasing cell–cell adhesion and endothelial integrity, endogenous Ang2 can facilitate the extravasation step at the endothelial cell–cell junctions. How Ang2 leads to the disruption of endothelial integrity remains to be elucidated. Such a mechanism may involve binding to integrins and loss of EC-EC junctional complexes, as reported for the related angiopoietin-like 4 (Angptl4) protein.
Although we show that the Ang2-blocking antibodies inhibit metastasis in immunodeficient mice in the absence of inflammatory cell contribution, Ang2 also affects the inflammatory and immune responses in tumors. Ang2 has been reported to stimulate Tie2-expressing monocytes to suppress T-cell activation and to promote regulatory T-cell expansion. In tumor-bearing immunocompetent mice, Ang2-blocking antibodies interfered with Tie2 expression in a subpopulation of macrophages and decreased angiogenesis, resulting in inhibition of metastasis. We found that the disruption of endothelial cell–cell and cell–matrix contacts in metastatic pulmonary capillaries was further aggravated in an immunocompetent background, suggesting that the immune response can contribute to endothelial destabilization.
This study had some limitations. We used rapidly growing tumors. Thus, the conclusions may be strengthened with the use of genetically engineered mouse tumor models. The dose–response range was not evaluated, and because of the rapid tumor growth and treatment schedules, possible adverse effects related to the treatment may have gone unnoticed. In addition, it remains to be investigated if the Ang2 antibodies can inhibit metastatic colonization of other tissues besides the lungs.
Our results are in line with the previously proposed model in which Ang2 becomes initially overexpressed in the tumor co-opted blood vessels, leading to vessel destabilization. This increases hypoxia in the tumor, and the expression of hypoxia-regulated VEGF and Ang2 growth factors, inducing robust tumor angiogenesis. The results from a study in which Ang2 was ectopically expressed in a mammary carcinoma cell line also support a model of dual action of Ang2 in the tumors. In that study, Ang2 expression induced intratumoral hemorrhage, non-functional and abnormal blood vessels, and was associated with pericyte loss and increased endothelial cell apoptosis, possibly because of imbalance of the Ang2 and VEGF levels in the tumors. It has been claimed that VEGFR2-targeted anti-angiogenic therapy increases metastatic frequency in some preclinical animal models. Our results showing that Ang2 induces vascular destabilization during tumor metastasis suggest that during VEGF blockade, Ang2-induced vascular destabilization could be associated with increased metastasis. Thus, combinatory therapy blocking both VEGF and Ang2 may provide more effective tumor growth and metastasis inhibition.
In conclusion, we show that Ang2 contributes substantially to tumor progression and that blocking of Ang2 inhibits endothelial destabilization and constitutes an efficient strategy for inhibition of tumor growth and metastatic dissemination. Our data thus provide important new insights into the Ang2/Tie2 signaling pathway as an attractive target for cancer therapy.
Discussion
Our results indicate that Ang2 increases tumor metastasis at least in part by promoting endothelial disruption and increasing tumor cell translocation and homing to target organs. We also provide mechanistic insight into the effects of the antibody-mediated blockade of Ang2 at the cellular level. To address the function of Ang2 in tumor metastasis, we used systemic and endothelial cell-specific Ang2 overexpression, as well as Ang2-blocking antibodies in various tumor models. Our results demonstrate that systemic Ang2 overexpression promotes tumor lymphangiogenesis as well as lymph node and lung metastasis in addition to tumor growth and angiogenesis. Ang2 also enhanced the establishment of metastatic colonies in the lungs after intravenous tumor cell inoculation. Importantly, by using VEC-tTA/Tet-OS-Ang2 transgenic mice, in which Ang2 is specifically induced only in the vascular endothelium, we demonstrated that endothelial overexpression of Ang2 increases lung metastasis. Alternatively, Ang2 blockade attenuated tumor lymphangiogenesis, dissemination of tumor cells via the lymphatic vessels, lung metastasis, and tumor cell colonization of the lungs.
The antiangiogenic effects of specific Ang2 blockade on tumor growth and vascularization have been previously characterized using Ang2 blocking in various tumor xenografts, but the effects on tumor metastasis were not investigated. In one report, intrapleural injection of lentiviral Ang2 short hairpin RNA in mice bearing mammary tumor xenografts was reported to inhibition endothelial permeability in the lungs. In another report, Ang2 overexpression in tumor cells increased their invasive properties by an autocrine, Tie2-independent effect mediated via the integrins. However, only a minor fraction of human tumor cell lines express detectable amounts of Ang2. Importantly, the lung cancer cells we used expressed no detectable Ang2 in culture, unlike in previous studies in which Ang2 inhibitors were used.
Targeted deletion of Ang2 leads to defective blood vessel development in the eye and lymphatic vessel development in the mesenterium and skin. In the developing postnatal retina, the Ang2-blocking antibody was found to delay vascularization, which was confirmed in our study. Further analysis showed that blocking Ang2 induced vascular tuft-like structures at the angiogenic front, where signs of sporadic sprout regression were observed. In the primary tumors treated with the Ang2-blocking antibody, basement membrane and pericyte marker–positive and endothelial cell negative vessel structures were present, similarly indicating endothelial regression. These results are consistent with the data that autocrine Ang2 contributes to endothelial cell survival and migration in some contexts (10,42, our unpublished results using Ang2-blocking antibody). In mice surviving Ang2 deletion, an initial delay was observed in the growth of isogenic tumors; however, in our study, the Ang2-blocking antibodies also induced statistically significant inhibition in the later phases of tumor growth. Thus, it is possible that genetic deletion of Ang2 and blocking antibodies against Ang2 have different mechanisms of action.
We and Fukuhara et al. have previously shown that angiopoietins induce the formation of unique Tie2 receptor complexes in endothelial cells. In cells with established contacts to other cells, Ang1 and Ang2 were found to trigger Tie2 translocation to cell–cell junctions, where Ang1 has been shown to reduce paracellular permeability. Here, we show that autocrine Ang2 secreted by endothelial cells similarly induces Tie2 and Tie1 translocation to cell–cell contacts. However, in contrast to Ang1, Ang2 induced only weak Tie2 phosphorylation and may thus decrease Ang1 signals via the Tie receptors in the endothelial cell–cell junctions. When the endothelial cells were cocultured with the tumor cells, the presence of Ang2-Tie2 complexes was increased in the endothelial cell–cell junctions, even when the cells were under normal oxygen pressure. The Ang2-blocking antibody inhibited endothelial Ang2 function by inducing the internalization of the Ang2-Tie2 receptor complexes and by inhibiting the subsequent formation of such complexes. Because of the multimeric nature of Ang2, the anti-Ang2 antibody may result in the formation of large receptor clusters on the cell surface; these could be more effectively targeted for endocytosis. The Ang1-induced Tie receptor complexes were not affected by the Ang2 antibody. Because only the Ang2-bound Tie2 receptor fraction was directed for internalization, the results suggest that during Ang2 blocking in the metastatic lungs, Ang1 can bind and activate Tie2, contributing to stabilization of endothelial cell–cell junctions.
Our results showed that fewer tumor cell colonies were established in the lungs of mice treated with the Ang2-blocking antibody when compared with control antibody–treated mice at the relatively early time points when angiogenesis was not yet detected in the metastatic foci. Although we have not imaged extravasation per se, these results suggest that blocking Ang2 inhibited the extravasation of tumor cells to the lungs or the very early stages of metastatic colony establishment. It was previously shown that the tumor cell–associated lung capillaries express increased amounts of Ang2. In the pulmonary vessels associated with the lung metastases, the Ang2-blocking antibody seemed to counteract the disruption of the endothelial cell–cell junctions and vascular integrity, suggesting that by decreasing cell–cell adhesion and endothelial integrity, endogenous Ang2 can facilitate the extravasation step at the endothelial cell–cell junctions. How Ang2 leads to the disruption of endothelial integrity remains to be elucidated. Such a mechanism may involve binding to integrins and loss of EC-EC junctional complexes, as reported for the related angiopoietin-like 4 (Angptl4) protein.
Although we show that the Ang2-blocking antibodies inhibit metastasis in immunodeficient mice in the absence of inflammatory cell contribution, Ang2 also affects the inflammatory and immune responses in tumors. Ang2 has been reported to stimulate Tie2-expressing monocytes to suppress T-cell activation and to promote regulatory T-cell expansion. In tumor-bearing immunocompetent mice, Ang2-blocking antibodies interfered with Tie2 expression in a subpopulation of macrophages and decreased angiogenesis, resulting in inhibition of metastasis. We found that the disruption of endothelial cell–cell and cell–matrix contacts in metastatic pulmonary capillaries was further aggravated in an immunocompetent background, suggesting that the immune response can contribute to endothelial destabilization.
This study had some limitations. We used rapidly growing tumors. Thus, the conclusions may be strengthened with the use of genetically engineered mouse tumor models. The dose–response range was not evaluated, and because of the rapid tumor growth and treatment schedules, possible adverse effects related to the treatment may have gone unnoticed. In addition, it remains to be investigated if the Ang2 antibodies can inhibit metastatic colonization of other tissues besides the lungs.
Our results are in line with the previously proposed model in which Ang2 becomes initially overexpressed in the tumor co-opted blood vessels, leading to vessel destabilization. This increases hypoxia in the tumor, and the expression of hypoxia-regulated VEGF and Ang2 growth factors, inducing robust tumor angiogenesis. The results from a study in which Ang2 was ectopically expressed in a mammary carcinoma cell line also support a model of dual action of Ang2 in the tumors. In that study, Ang2 expression induced intratumoral hemorrhage, non-functional and abnormal blood vessels, and was associated with pericyte loss and increased endothelial cell apoptosis, possibly because of imbalance of the Ang2 and VEGF levels in the tumors. It has been claimed that VEGFR2-targeted anti-angiogenic therapy increases metastatic frequency in some preclinical animal models. Our results showing that Ang2 induces vascular destabilization during tumor metastasis suggest that during VEGF blockade, Ang2-induced vascular destabilization could be associated with increased metastasis. Thus, combinatory therapy blocking both VEGF and Ang2 may provide more effective tumor growth and metastasis inhibition.
In conclusion, we show that Ang2 contributes substantially to tumor progression and that blocking of Ang2 inhibits endothelial destabilization and constitutes an efficient strategy for inhibition of tumor growth and metastatic dissemination. Our data thus provide important new insights into the Ang2/Tie2 signaling pathway as an attractive target for cancer therapy.