Lack of Angiogenesis in the Formation of Lymph Node Metastases
Lack of Angiogenesis in the Formation of Lymph Node Metastases
The main concept driving antiangiogenic therapy has been the hypothesis that tumors depend on new blood vessel growth. A critical observation made by longitudinal intravital microscopy in the CLNW is that metastatic lesions did not induce sprouting angiogenesis as they grew, in spite of the presence of hypoxia. Lesions that invaded into the blood vessel–rich lymph node parenchyma showed reduced hypoxia, suggesting that cancer cells survive in the lymph node by utilizing the existing lymph node vascular supply. The lack of VEGF, VEGF-C, and VEGF-D, along with the presence of TSP-1 surrounding lymph node blood vessels, provides a mechanism behind the lack of sprouting angiogenesis observed in lymph node metastases. A limitation of the use of longitudinal intravital microscopy is the limited imaging depth of 300 μm by multiphoton microscopy in the CLNW. To balance this, we used histological techniques, which allow full lymph node depth to be characterized but are limited in their ability to monitor the kinetic changes occurring as metastatic lesions grow in the lymph node. Using these complimentary techniques allowed better characterization of the growth of lymphatic metastases.
Our data show lymph node lymphangiogenesis is an early event in the natural history of cancer progression, in agreement with previous studies. However, decreased lymphatic vessel density was found in macrometastatic lymph nodes, suggesting that the presence of the cancer cells in the lymph node causes lymphatic vessel regression. Furthermore, bevacizumab did not statistically significantly affect the lymphatic vasculature in patients. These data suggest that late intervention with antiangiogenic or antilymphangiogenic therapies after lymphatic vessel regression has begun in patients will show no effect on lymph node lymphatic vessels.
In patients, the observation that large metastatic lesions do not exhibit increased vascular density relative to those with micrometastases further suggests that sprouting angiogenesis is not required to sustain the growth of lymph node metastases. A limitation of our data is that we estimated lesion size based on the two-dimensional area available in the histological sections, so we are likely underestimating the size of the lesion. An additional limitation of our study is that we cannot rule out the contribution from different modes of new blood vessel formation in lymph node metastasis such as vasculogenesis, intussusception, vessel co-option, vascular mimicry, and tumor cell differentiation into endothelial cells. The mechanisms of these alternative processes are not clearly defined, although VEGF and endothelial proliferation have been shown to contribute to these processes. Our preclinical and clinical data, however, show that inhibitors targeting primarily sprouting angiogenesis will not inhibit the growth of metastases in the lymph node.
Predicted by recent genomic data, we provide direct evidence that lymph node metastasis forms from multiple cells that disseminate from the primary tumor and suggest a fundamental difference in their formation compared with hematogenous metastases. Cancer cells that invade lymphatic vessels travel to the draining lymph node where they enter in locations defined by afferent lymphatic vessels. As such, lymph node metastasis can be reinforced by the continual arrival of new cells as they gain a foothold in their new microenvironment, leading to the spatially heterogeneous lesions imaged here and the genetically heterogeneous lesions documented previously. In contrast, cells that metastasize through the blood spread out to different locations in an organ by the branching vasculature, leading to a higher probability of individually homogenous lesions. One can thus speculate that targeting a single genetic trait, unless ubiquitous in the primary tumor, may not be effective in eradicating lymph node metastases and any subsequent spread to distant sites.
Using multiple spontaneous metastasis models, we show the first direct evidence that sprouting angiogenesis is not required in lymph node lesions during early metastatic growth. The lack of sprouting angiogenesis in lymph node metastases suggests an additional explanation for the poor outcomes of antiangiogenic therapy in adjuvant settings. As the lymph node is able to metabolically support rapid cellular expansion during an active immune response, it seems the existing vasculature of the lymph node is also able to support the growth of a nascent metastasis. Thus, the mechanisms of angiogenesis and the targets of clinically approved drugs are not active during this early step in cancer progression, suggesting that inhibitors of sprouting angiogenesis as a class will not be effective in treating lymph node metastases. Our novel preclinical models provide opportunities to uncover strategies to better control and eradicate disease in lymph nodes in metastatic cancer patients.
Discussion
The main concept driving antiangiogenic therapy has been the hypothesis that tumors depend on new blood vessel growth. A critical observation made by longitudinal intravital microscopy in the CLNW is that metastatic lesions did not induce sprouting angiogenesis as they grew, in spite of the presence of hypoxia. Lesions that invaded into the blood vessel–rich lymph node parenchyma showed reduced hypoxia, suggesting that cancer cells survive in the lymph node by utilizing the existing lymph node vascular supply. The lack of VEGF, VEGF-C, and VEGF-D, along with the presence of TSP-1 surrounding lymph node blood vessels, provides a mechanism behind the lack of sprouting angiogenesis observed in lymph node metastases. A limitation of the use of longitudinal intravital microscopy is the limited imaging depth of 300 μm by multiphoton microscopy in the CLNW. To balance this, we used histological techniques, which allow full lymph node depth to be characterized but are limited in their ability to monitor the kinetic changes occurring as metastatic lesions grow in the lymph node. Using these complimentary techniques allowed better characterization of the growth of lymphatic metastases.
Our data show lymph node lymphangiogenesis is an early event in the natural history of cancer progression, in agreement with previous studies. However, decreased lymphatic vessel density was found in macrometastatic lymph nodes, suggesting that the presence of the cancer cells in the lymph node causes lymphatic vessel regression. Furthermore, bevacizumab did not statistically significantly affect the lymphatic vasculature in patients. These data suggest that late intervention with antiangiogenic or antilymphangiogenic therapies after lymphatic vessel regression has begun in patients will show no effect on lymph node lymphatic vessels.
In patients, the observation that large metastatic lesions do not exhibit increased vascular density relative to those with micrometastases further suggests that sprouting angiogenesis is not required to sustain the growth of lymph node metastases. A limitation of our data is that we estimated lesion size based on the two-dimensional area available in the histological sections, so we are likely underestimating the size of the lesion. An additional limitation of our study is that we cannot rule out the contribution from different modes of new blood vessel formation in lymph node metastasis such as vasculogenesis, intussusception, vessel co-option, vascular mimicry, and tumor cell differentiation into endothelial cells. The mechanisms of these alternative processes are not clearly defined, although VEGF and endothelial proliferation have been shown to contribute to these processes. Our preclinical and clinical data, however, show that inhibitors targeting primarily sprouting angiogenesis will not inhibit the growth of metastases in the lymph node.
Predicted by recent genomic data, we provide direct evidence that lymph node metastasis forms from multiple cells that disseminate from the primary tumor and suggest a fundamental difference in their formation compared with hematogenous metastases. Cancer cells that invade lymphatic vessels travel to the draining lymph node where they enter in locations defined by afferent lymphatic vessels. As such, lymph node metastasis can be reinforced by the continual arrival of new cells as they gain a foothold in their new microenvironment, leading to the spatially heterogeneous lesions imaged here and the genetically heterogeneous lesions documented previously. In contrast, cells that metastasize through the blood spread out to different locations in an organ by the branching vasculature, leading to a higher probability of individually homogenous lesions. One can thus speculate that targeting a single genetic trait, unless ubiquitous in the primary tumor, may not be effective in eradicating lymph node metastases and any subsequent spread to distant sites.
Using multiple spontaneous metastasis models, we show the first direct evidence that sprouting angiogenesis is not required in lymph node lesions during early metastatic growth. The lack of sprouting angiogenesis in lymph node metastases suggests an additional explanation for the poor outcomes of antiangiogenic therapy in adjuvant settings. As the lymph node is able to metabolically support rapid cellular expansion during an active immune response, it seems the existing vasculature of the lymph node is also able to support the growth of a nascent metastasis. Thus, the mechanisms of angiogenesis and the targets of clinically approved drugs are not active during this early step in cancer progression, suggesting that inhibitors of sprouting angiogenesis as a class will not be effective in treating lymph node metastases. Our novel preclinical models provide opportunities to uncover strategies to better control and eradicate disease in lymph nodes in metastatic cancer patients.
