Metastasis of breast cancer occurs primarily through the lymphatic system, and the extent of lymph node involvement is a key prognostic factor for the disease. Whereas the significance of angiogenesis for tumor progression has been well documented, the ability of tumor cells to induce the growth of lymphatic vessels (lymphangiogenesis) and the presence of intratumoral lymphatic vessels have been controversial. Using a novel marker for lymphatic endothelium, LYVE-1, we demonstrate here the occurrence of intratumoral lymphangiogenesis within human breast cancers after orthotopic transplantation onto nude mice. Vascular endothelial growth factor (VEGF)-C overexpression in breast cancer cells potently increased intratumoral lymphangiogenesis, resulting in significantly enhanced metastasis to regional lymph nodes and to lungs. The degree of tumor lymphangiogenesis was highly correlated with the extent of lymph node and lung metastases. These results establish the occurrence and biological significance of intratumoral lymphangiogenesis in breast cancer and identify VEGF-C as a molecular link between tumor lymphangiogenesis and metastasis.

Acquisition of invasive/metastatic potential through protease expression is an essential event in tumor progression. High levels of components of the plasminogen activation system, including urokinase, but paradoxically also its inhibitor, plasminogen activator inhibitor 1 (PAI1), have been correlated with a poor prognosis for some cancers. We report here that deficient PAI1 expression in host mice prevented local invasion and tumor vascularization of transplanted malignant keratinocytes. When this PAI1 deficiency was circumvented by intravenous injection of a replication-defective adenoviral vector expressing human PAI1, invasion and associated angio-genesis were restored. This experimental evidence demonstrates that host-produced PAI is essential for cancer cell invasion and angiogenesis.Tumor cell invasion and metastatic processes require the coordinated and temporal regulation of a series of adhesive, proteolytic and migratory events 1 . The plasminogen activator (PA)-plasmin proteolytic system has been implicated in these processes. Urokinase-type (uPA) and tissue-type (tPA) plasminogen activators are serine proteases that catalyze the conversion of inactive plasminogen into plasmin, a broadly acting enzyme able to degrade a variety of extracellular matrix proteins and to activate metalloproteinases and growth factors 2,3 . Plasminogen and uPA bind to their specific receptors directing plasmin activity to the migrating tumor cell surface. The activities of PA are directly controlled by specific inhibitors, the PA inhibitors 1 and 2 (PAI1 and PAI2) (ref. 4).Many studies have focused on the role of uPA in cellular invasion and metastasis. Much of the data supporting the role of uPA in these events derives from in vitro and in vivo experiments demonstrating a correlation between uPA expression and cell invasion and metastasis as well as reduction of metastatic potential by using natural or synthetic serine protease inhibitors, neutralizing antibodies to uPA or antisense oligonucleotides 5,6 . PAI1 may also be directly involved in cancer progression. Both tumor cells and capillary endothelial cells express higher levels of PAI1 than other cell types [7][8][9] . Surprisingly, this inhibitor is necessary for optimal invasion of cultured lung cancer cells 10 , and an increasing number of clinical studies have demonstrated that high PAI1 levels indicate a poor prognosis for the survival of patients suffering from a variety of cancers [11][12][13] . However, as PAI1 is an acute-phase reactant 14 , it remains undetermined whether the increased PAI1 levels causally contribute to, or rather are the consequence of, the malignancy.Various observations indicate that the PA system may provide both surface-associated protease activity and an adhesion mechanism for cells through interaction with vitronectin. Deng et al. suggested that the balance between cell adhesion and cell detachment is governed by PAI1 (ref. 15). The PAI1-mediated release of cells attached to vitronectin seems to occur independently of the abili...

was inadvertently switched during the revision process with a previously published figure (1) showing results on B16 melanoma cells. The correct figure, summarizing the results on human MeWO melanoma cells transfected with ␣1,3-fucosyltransferase (FTIII), appears below. We apologize for any confusion caused by this mistake.

The lymphatic system transports interstitial fluid and macromolecules from tissues back to the blood circulation, and plays an important role in the immune response by directing the traffic of lymphocytes and antigen-presenting cells. The lymphatic system also constitutes one of the most important pathways of tumor dissemination. In many human cancers, increased expression of vascular endothelial growth factor-C (VEGF-C) is correlated with regional lymph node metastases. Experimental studies using transgenic mice overexpressing VEGF-C or xenotransplantation of VEGF-C-expressing tumor cells into immunodeficient mice have demonstrated a role for VEGF-C in tumor lymphangiogenesis and the subsequent formation of lymph node metastases. However, there is at present little evidence for lymphangiogenesis in human tumors and the relative importance of preexisting vs. newly formed lymphatics for metastasis in humans remains to be determined. Nonetheless, the striking correlation between the levels of VEGF-C in primary human tumors and lymph node metastases predicts its importance in cancer spread. Aside from promoting lymphangiogenesis, VEGF-C may also activate lymphatics to promote tumor cell chemotaxis, lymphatic intravasation and hence tumor cell dissemination.

Interactions of tumor cells with lymphatic vessels are of paramount importance for tumor progression, however, the underlying molecular mechanisms are poorly understood. Whereas enlarged lymphatic vessels are frequently observed at the periphery of malignant melanomas, it has remained unclear whether intratumoral lymphangiogenesis occurs within these tumors. Here, we demonstrate the presence of intratumoral lymphatics and enlargement of lymphatic vessels at the tumor periphery in vascular endothelial growth factor (VEGF)-C-overexpressing human melanomas transplanted onto nude mice. VEGF-C expression also resulted in enhanced tumor angiogenesis, indicating a coordinated regulation of lymphangiogenesis and angiogenesis in melanoma progression. The specific biological effects of VEGF-C were critically dependent on its proteolytic processing in vivo.

Immunity 24, 203-215; February 2006) The text related to Figure 2F in the Results section of our paper conveys an incorrect calculation that affects the interpretation of the experiments in Figure 2F, although not the overall conclusion we drew. The text as stated indicates that very few DCs entered efferent lymph to arrive at a lymph node (LN) further downstream. However, this extremely low magnitude (0.01%) is incorrect because it was mistakenly derived from the total fraction of LN cells, not the fraction of migrating, labeled DCs. The correct value, as is evident from the figure itself, is that 12% of traceable DCs traversed a primary draining lymph node (popliteal) to enter the downstream iliac lymph node (397 6 84 labeled DCs in the popliteal LN versus 49 6 21 in the iliac LN). This value was not affected by immunization that induced inflammation in the popliteal LN, as originally stated. Although these data indicate that DCs enter efferent lymph to a reasonable extent, the magnitude is far too low to account for the difference in the number of DCs found in LNs that primarily drain nonimmunized and immunized LNs (such as in Figure 1C).

Lymph nodes are the first site of metastases for most types of cancer, and lymph node status is a key indicator of patient prognosis. Induction of tumor lymphangiogenesis by vascular endothelial growth factor-C (VEGF-C) has been shown to play an important role in promoting tumor metastases to lymph nodes. Here, we employed receptor-specific antagonist antibodies in an orthotopic spontaneous breast cancer metastasis model to provide direct evidence for the key role of VEGFR-3 activation in metastasis. Inhibition of VEGFR-3 activation more potently suppressed regional and distant metastases than inactivation of VEGFR-2, although VEGFR-2 blockade was more effective in inhibiting angiogenesis and tumor growth. Despite prominent proliferation, metastases were not vascularized in any of the control and treatment groups, indicating that the growth of metastases was not dependent on angiogenesis at the secondary site for the duration of the experiment. Systemic treatment with either VEGFR-2 or VEGFR-3 antagonistic antibodies suppressed tumor lymphangiogenesis, indicating that VEGFR-3 signaling affects the rate of tumor cell entry into lymphatic vessels through both lymphangiogenesis-dependent and independent mechanisms. Combination treatment with the anti-VEGFR-2 and anti-VEGFR-3 antibodies more potently decreased lymph node and lung metastases than each antibody alone. These results validate the concept of targeting the lymphatic dissemination and thereby very early steps of the metastatic process for metastasis control and suggest that a combination therapy with antiangiogenic agents may be a particularly promising approach for controlling metastases. (Cancer Res 2006; 66(5): 2650-7)

Dendritic cell (DC) migration from the periphery to lymph nodes is regulated by the pattern of genes expressed by DCs themselves and by signals within the surrounding peripheral environment. Here, we report that DC mobilization can also be regulated by signals initiated within the downstream lymph nodes, particularly when lymph nodes enlarge as a consequence of immunization. Lymph node B lymphocytes orchestrate expansion of the lymphatic network within the immunized lymph node. This expanded network in turn supports increased DC migration from the periphery. These results reveal unique relationships between B cells, lymphatic vessels, and migratory DCs. Knowledge that DC migration from the periphery is augmented by B cell-dependent signals reveals new potential strategies to increase DC migration during vaccination.