Lymphangiogenesis and cancer metastasis.

Proc Amer Assoc Cancer Res, Volume 47, 2006 SY11-01 The induction of angiogenesis and neovascularization is an important mechanism by which tumors promote their continued growth and subsequent metastasis. The lymphatic vascular system offers another particularly apt conduit for the spread of tumor cells, as this network of vessels functions naturally as an exit route for immune cells from tissues. Indeed, the propensity for lymphatic metastasis is a common feature of most solid tumors. In contrast to tumors that are composed of heterogenous and adaptive malignant cells, which easily evolve to become resistant to treatment, blood and lymphatic endothelial cells represent normal diploid cells and they should only grow in response to growth factors secreted by tumor cells. Recent successes in the treatment of colorectal cancer patients with monoclonal neutralizing antibodies to vascular endothelial growth factor (VEGF) have demonstrated the potential of highly specific molecular therapies targeting endothelial cells and the angiogenic process. Similar approaches could eventually be used to target lymphatic vessels for the development of anti-metastatic therapies. Despite the importance of the lymphatic vasculature very little was known, until recently, about the mechanisms of lymph vessel growth and development. Lymphatic vessels were first described in the 17th century and their development described a century ago. However it was only 10 years ago that any growth factors and molecular markers specific to lymph vessels were discovered, sparking intense activity in the field. The lymphatic vasculature is essential for the maintenance of interstitial fluid balance, for immune defence and for the uptake of dietary fat. Lymphatic aplasia, hypoplasia or obstructive, obliterative or surgical hindrance of lymph drainage causes stagnation of proteins and associated water in the interstitium, and leads to lymphedema, a disfiguring and disabling swelling of the extremities. Dissemination of tumor cells via lymphatic vessels to distant organs is a leading cause of death in cancer patients, and a major obstacle in the design of effective therapies. Even though metastasis via the lymph is common in cancers, little attention has been paid to the lymphatic vasculature in tumors and the mechanisms regulating its development and growth. The situation has changed during the past few years, mainly because of progress in the identification of regulatory molecules and markers specific for the lymphatic endothelial cells. Lymphangiogenesis accompanies angiogenesis in both embryogenesis and disease, yet the morphologic and functional differences between lymphatic and blood capillaries are striking. The basis for these differences may lie in the genes expressed by lymphatic and blood endothelial cells. Lymphatic endothelial cell differentiation commitment and growth during the embryonic development are dependent on the activities of the homeodomain transcription factor Prox-1 and VEGF-C, respectively. Comparison of the transcriptional profiles of blood vessel and lymphatic vessel endothelial cells on a genome-wide scale has opened the possibility to systematically analyze differences between these two cell lineages. The transcripts expressed by lymphatic endothelial cells and blood vascular endothelial cells are modified soon after their isolation from tissues and approximately 2% of transcribed genes are differentially expressed between these two cell types. For example, blood vascular endothelial cells express significantly higher levels of pro-inflammatory cytokines, chemokines and chemokine receptors, have higher levels of β-catenin and display increased numbers of actin stress fibers. The genetic programs that determine the complexity of lymphatic capillaries and the larger collecting lymphatic vessels and make them distinct from blood vessels involve several other newly described signal transduction pathways. For example, the FOXC2 transcription factor has been implicated in the formation of the lymph vessels and their valves. As VEGF-C and VEGF-D induce adult lymphangiogenesis, associated with inflammation, wound healing and tumor metastasis, administration of lymphatic growth factors or their antagonists could provide the opportunity to target lymphatic vessels in human disease. Five members of the human VEGF family interact with the VEGF receptors (VEGFRs) to relay the intracellular signals responsible for the induction of growth, migration and survival of endothelial cells. In addition, the VEGFs interact with neuropilin coreceptors. In the lymphatic vessels VEGF-C and VEGF-D interact with VEGFR-3 and neuropilin-2. Neuropilin-2 also serves as a plexin co-receptor for type III semaphorins in the nervous system. Although all three VEGFRs are required for the development of blood vessels in embryos, VEGFR-3 expression becomes largely confined to the lymphatic endothelium in adults. Additional, less explored signal transduction pathways include the interaction of VEGF-C with integrin α9, and VEGFR-3 with integrin β1 and human Kaposi sarcoma herpesvirus-8 envelope protein gB. After proteolytic cleavage of the N- and C-terminal propeptides of VEGF-C and VEGF-D these proteins acquire the ability to activate VEGFR-2, also present in the lymphatic endothelium. Correspondingly, the processed forms of VEGF-C and VEGF-D are able to induce angiogenesis via the VEGFR-2 on blood vessels. Although studies have clearly shown that lymphangiogenesis is induced by VEGF-C and VEGF-D via VEGFR-3 activation, recent findings also suggest that VEGF, angiopoietin-1, and fibroblast, hepatocyte and insulin-like growth factors may also mediate lymphangiogenesis directly or indirectly. In most cancers, the metastatic spread of tumor cells to distant organs results in patient mortality. Tumor metastasis occurs to lymph nodes and distant organs via lymph or blood vessels. The patterns of metastasis vary from tumor to tumor, but regional lymph nodes are often the first site of metastasis in carcinomas. Metastatic nodes are important in the staging, treatment and follow-up of many solid tumors. Regional lymph node metastasis is often the most important prognostic factor for patients with malignant tumors of epithelial origin. The sentinel lymph node is the first regional lymph node to be invaded by tumor cells; further metastasis may then occur to other nodes and to the systemic circulation. The process of metastasis is complex and involves changes in the expression of many genes. In most tumors, it is not known if metastasis via the lymph selects for cells with high affinity for a distant destination, or if the appearance of lymphatic metastasis just indicates that the tumor has entered a metastatic phase in general, including organ metastasis. Growth factor stimulation of lymphatic vessels appears to enhance lymphatic metastasis, but many questions about the mechanisms of lymphatic dissemination remain unanswered. VEGF-C and VEGF-D expression correlate with vascular invasion, lymphatic vessel and lymph node involvement, distant metastasis, and, in some instances, poor clinical outcomes. Also studies of various tumor models have shown that VEGF-C and VEGF-D overexpression can enhance lymphatic metastasis. Furthermore, a soluble VEGFR-3 fusion protein (“VEGF-C/D Trap”) inhibited lymphatic metastasis in several tumor models. In some models lymphatic, but not lung metastases were blocked with a VEGF-C/D trap while in others the treatment inhibited both lymph node and lung metastases. Although these experiments provide strong support for the involvement of VEGF-C, VEGF-D, and their receptor VEGFR-3 in the lymphatic spread of malignancy, the underlying mechanisms have only recently been addressed. Lymph vessel proliferation seen in tumor models overexpressing the lymphangiogenic factors may not be a prominent feature in several human cancers, and may in fact not be needed for enhanced metastasis in most solid tumors. While intratumoral lymphatic vessels have been detected in some solid tumors, such as melanomas and head and neck carcinomas, at least in experimental tumors they may not be completely functional, because of their collapse in conditions of high intratumoral pressure. On the other hand, the pressure gradient and lymph vessels at the tumor margin may be more important in spreading tumor cells through the process of vessel sprouting stimulated by tumor-secreted VEGF-C or VEGF-D. In this process the endothelial cells send long filopodia towards the growth factor producing tumor cells and then form tumor-directed vessel sprouts, where the vessel lumen opens up and may allow facilitated access of tumor cells to the lumen. In some cases the lymphatic endothelium could also actively participate in metastasis formation by secreting chemokines that have receptors in tumor cells. Furthermore, the collecting lymphatic vessels draining fluid from the tumor area are stimulated to dilate by intraluminal VEGF-C via the process of endothelial proliferation in the vessel wall. Clumps of metastatic tumor cells could then undergo an easier transit in lymph flowing in the dilated hyperplastic vessels. The VEGF-C/D trap inhibited sprouting and vessel dilation, and seemed to restore the integrity of the vessel wall. Similarly, blocking monoclonal antibodies that target VEGF-C, VEGF-D or their receptor(s) and small molecules that inhibit the tyrosine kinase catalytic domain of these receptors could be used for the inhibition of experimental tumor metastasis. Further work should soon tell if these same molecules inhibit further systemic metastasis or angiogenesis in tumor models.