Lymphangiogenesis and lymphatic vessel remodelling in cancer

Journal name:
Nature Reviews Cancer
Volume:
14,
Pages:
159–172
Year published:
DOI:
doi:10.1038/nrc3677
Published online

Abstract

The generation of new lymphatic vessels through lymphangiogenesis and the remodelling of existing lymphatics are thought to be important steps in cancer metastasis. The past decade has been exciting in terms of research into the molecular and cellular biology of lymphatic vessels in cancer, and it has been shown that the molecular control of tumour lymphangiogenesis has similarities to that of tumour angiogenesis. Nevertheless, there are significant mechanistic differences between these biological processes. We are now developing a greater understanding of the specific roles of distinct lymphatic vessel subtypes in cancer, and this provides opportunities to improve diagnostic and therapeutic approaches that aim to restrict the progression of cancer.

At a glance

Figures

  1. Structure of the lymphatic vasculature and of lymphatic endothelial cells from initial lymphatics.
    Figure 1: Structure of the lymphatic vasculature and of lymphatic endothelial cells from initial lymphatics.

    The hierarchy of lymphatic vessel subtypes is shown in part a, with the characteristics of the subtypes indicated. The association between lymphatic endothelial cells (LECs) in initial lymphatics is shown in the two pull-out boxes, with the contact regions between LECs in the vessels being indicated by the brown lines in the first pull-out box. Fluid is thought to enter (as shown by the arrows in the second pull-out box) via the tips of the flaps of LECs, without compromising the integrity of the intercellular junctions on the sides of the flaps13. The structure of LECs from initial lymphatics is shown in part b, illustrating the entry of fluid and cells, which is favoured when the interstitial fluid pressure is high, causing stretching of the extracellular matrix. The structure of a blood vascular capillary is shown for comparison. The anchoring filaments that are associated with initial lymphatic vessels connect the abluminal membrane of LECs to surrounding elastic fibres — these filaments are short, but for visual clarity they are shown larger than to scale. Anchoring filaments are involved in controlling the entry of cells and interstitial fluid into the initial lymphatics. It should be noted that cells can cross lymphatic endothelial layers via a transcellular route, as well as via the paracellular route that is shown here. BEC, blood vascular endothelial cell; BM, basement membrane; SMC, smooth muscle cell.

  2. Remodelling of lymphatic vessels in cancer and its contribution to metastasis.
    Figure 2: Remodelling of lymphatic vessels in cancer and its contribution to metastasis.

    Lymphatic vessels that are evident in adults arise during embryonic development. In cancer, these pre-existing lymphatics undergo various types of remodelling, including lymphangiogenesis (resulting in the generation of new lymphatics) and lymphatic enlargement. Lymphatic enlargement does not generate new vessels and can involve proliferative or non-proliferative mechanisms. Lymphangiogenic growth factors, which are derived from tumour cells or immune cells in the tumour microenvironment, promote tumour lymphangiogenesis and/or lymphatic enlargement in and around the primary tumour. These proteins also promote lymph node lymphangiogenesis, enlargement of tumour-draining collecting lymphatics (both proximal and distal to sentinel lymph nodes (SLNs; identified using a tracer)) and alterations to lymphatic smooth muscle cells (LSMCs) around collecting lymphatics6, 7, 8. These processes are thought to facilitate metastasis. The mechanism of transit of tumour cells from lymph nodes to distant organs is currently speculative.

  3. Mechanisms of tumour lymphangiogenesis and interactions between tumour cells and lymphatics.
    Figure 3: Mechanisms of tumour lymphangiogenesis and interactions between tumour cells and lymphatics.

    Molecules that are reported to modulate tumour lymphangiogenesis are shown, with soluble ligands presented outside the cell, cognate receptors at the cell surface and transcription factors in the nucleus. Vascular endothelial growth factor C (VEGFC) and VEGFD refer to the proteolytically processed, biologically active forms of these proteins. Most of the ligands shown have been reported to promote lymphangiogenesis, whereas transforming growth factor-β (TGFβ) is considered to be an inhibitor of lymphangiogenesis. There are other molecules that are known to participate in lymphatic development in the embryo, such as collagen and calcium binding EGF domain-containing protein 1 (CCBE1; not shown), for which a role in tumour lymphangiogenesis has not been shown. The interaction of tumour cells with lymphatic vessels can be promoted by interstitial fluid flow (which partly results from lymphatic drainage) via autologous chemotaxis98 that involves chemokines, such as CC-chemokine ligand 21 (CCL21), and their receptors (CCR7 in the case of CCL21), which are expressed by tumour cells. Expression of CCL21 on lymphatic endothelial cells (LECs) can promote the entry of tumour cells into lymphatics via a CCR7-dependent mechanism. Production of lymphangiogenic growth factors, such as VEGFC and VEGFD, can drive the formation of new lymphatics and lymphatic enlargement in the vicinity of a tumour, which increases the surface area for the interaction of tumour cells with lymphatics. VEGFC can also promote tumour cell invasiveness in an autocrine manner, and it can upregulate the production of CCL21 on lymphatic vessels100. 15-PGDH, 15-hydroxyprostaglandin dehydrogenase; ANGPT2, angiopoietin 2; COUPTF2, COUP transcription factor 2; COX2, cyclooxygenase 2; CSF1, colony-stimulating factor 1; EGF, epidermal growth factor; EGFR, EGF receptor; EPO, erythropoietin; EPOR, EPO receptor; FGF, fibroblast growth factor; FGFR, FGF receptor; FOXC2, forkhead box protein C2; PDGF-BB, platelet-derived growth factor BB; PDGFR, PDGF receptor; PROX1, prospero homeobox protein 1; RAMP2, receptor activity-modifying protein 2; S1P, sphingosine-1-phosphate; TGFβR, TGFβ receptor; VEGFR, VEGF receptor.

  4. Lymphangiogenesis and lymphatic remodelling in cancer [mdash] implications for diagnostics.
    Figure 4: Lymphangiogenesis and lymphatic remodelling in cancer — implications for diagnostics.

    Using breast cancer as an example, the anatomical features affected by lymphangiogenesis and lymphatic remodelling in cancer are shown in the boxes in bold; the boxes also show the changes that occur to these features in cancer and the actual or potential diagnostic approaches for monitoring these changes. ELISA, enzyme-linked immunosorbent assay; I-PET, immuno-positron emission tomography.

Key points

  • The lymphatic vasculature is essential for immune function, tissue fluid homeostasis and the absorption of dietary fat.
  • The process of lymphangiogenesis involves the formation of new lymphatic vessels from pre-existing lymphatics; this occurs during embryonic development, wound healing and in various pathological contexts, including cancer.
  • Tumour cells and cells of the tumour microenvironment produce growth factors that promote lymphangiogenesis from initial lymphatics, as well as the enlargement of initial and collecting lymphatic vessels in and around solid tumours. The enlargement of collecting lymphatics can involve remodelling of these vessels by smooth muscle cells.
  • Lymphangiogenic factors (such as vascular endothelial growth factor C (VEGFC) and VEGFD) can induce the metastatic spread of tumours in mouse models of cancer.
  • Clinicopathological studies have shown that the production of lymphangiogenic factors, lymphangiogenesis and lymphatic remodelling can correlate with cancer progression.
  • Lymphatic vessels provide a therapeutic target for modulating the immune response to cancer and restricting metastasis; clinical trials of agents that target lymphangiogenic signalling pathways are underway.
  • Mouse models and genome-wide functional screening approaches might identify further important signalling pathways in tumour lymphangiogenesis that could be potential diagnostic and therapeutic targets.

Introduction

The metastasis of tumour cells is a leading cause of mortality in cancer patients and occurs through lymphatic vessels and blood vessels. The presence of tumour cells in regional or sentinel lymph nodes (SLNs) is a key predictor of poor outcome in human cancer1, which illustrates the relevance of lymphatics to cancer biology. It was previously thought that the lymphatic vasculature had a passive role in cancer metastasis; however, experimental and clinicopathological studies indicate that lymphatic vessels undergo dynamic changes that facilitate metastasis. These changes include lymphangiogenesis and lymphatic enlargement (which can occur through lymphatic hyperplasia), and they involve initial lymphatics around the primary tumour. These events are thought to favour entry of tumour cells into the lymphatic vasculature2, 3, 4, 5. The enlargement of collecting lymphatics that are proximal or distal to SLNs, which is a process that can involve proliferation of lymphatic endothelial cells (LECs), other non-proliferative mechanisms and alterations to lymphatic-associated vascular smooth muscle cells (LVSMCs), has also been reported and is thought to facilitate the dissemination of tumour cells6, 7. Moreover, lymphangiogenesis in tumour-draining lymph nodes might increase the spread of tumour cells to distant sites in the body8.

The recognition that lymphangiogenesis and lymphatic remodelling are functionally important in cancer has led to the idea that blocking them, by targeting lymphangiogenic signalling pathways, might be a useful therapeutic strategy to restrict metastatic spread9. Furthermore, the remodelling of tumour-associated lymphatics and of lymphatics in tumour-draining lymph nodes might provide prognostic opportunities10. In this Review we discuss lymphangiogenesis and lymphatic remodelling in cancer, and we focus on recent progress in defining the molecular and cellular mechanisms that control these processes. The implications of these findings for the development of new diagnostics and therapeutics, and for future cancer research, are also discussed.

Structure of the lymphatic system

Although the structure and physiology of the lymphatic system have been studied since the Renaissance11, advances in identifying molecular markers of lymphatics (Box 1), which have predominantly occurred over the past 20 years, have focused on the unique cellular and functional features of this system and its role in pathology. The lymphatic vasculature is essential for immune function, tissue fluid homeostasis and for the absorption of dietary fat. Starting as small blind-ended vessels that absorb fluid and cells in tissues12, 13, the initial lymphatics are characterized by short anchoring filaments, which connect the abluminal membrane of LECs to the surrounding elastic fibres in the tissue12 (Fig. 1). These lymphatics have an intermittent basement membrane and no pericytes or VSMCs. The tethering of LECs to elastic fibres, as well as unique discontinuous 'button-like' cell–cell junctions between LECs allow flaps of the vessel to open and fluid to enter but not to leave. The anchoring filaments are particularly important when the interstitial fluid pressure is increased (as occurs in oedema), because this stretches the extracellular matrix (ECM) and the elastic fibres and thereby further opens the initial lymphatics to enable interstitial fluid drainage13. Fluid and cells can also enter these vessels through a transcellular route14. Fluid is then fed into deeper pre-collecting vessels, then into larger calibre lymphatic collecting vessels (which are distinguished by the presence of a basement membrane, valves to regulate flow and a surrounding layer of VSMCs) and then back into the bloodstream through the thoracic duct15, 16, 17 (Fig. 1). The lymph fluid passes through lymph nodes when transiting through the collecting lymphatics. In cancer patients, the different types of lymphatic vessels can be influenced by tumour-derived growth factors, which leads to lymphangiogenesis, other types of lymphatic remodelling and to the modulation of immune function, all of which can increase the metastatic spread of tumour cells to lymph nodes and potentially to distant organs (see below and Fig. 2).

Box 1: Markers of tumour lymphatic vessels

Various proteins have been identified that are expressed on lymphatic vessels, including prospero homeobox protein 1 (PROX1), SOX18, COUP transcription factor 2 (COUPTF2), forkhead box protein C2 (FOXC2), neuropilin 2 (NRP2), vascular endothelial growth factor receptor 3 (VEGFR3) and others, some of which have important functions in the development of the lymphatic vasculature (reviewed in Refs 16,32). However, over the past 5 years, only two of these proteins have been routinely monitored in cancer to identify lymphatic vessels: lymphatic vessel endothelial hyaluronic acid receptor 1 (LYVE1)145 and podoplanin146. Antibodies to these proteins have been used for immunohistochemistry or immunofluorescence to distinguish lymphatic vessels from blood vessels in human tumours and in experimental tumours in mice. LYVE1 is a CD44 homologue that is involved in hyaluronic acid transport145 and that is generally expressed on lymphatic endothelial cells (LECs) of small lymphatic vessels but not on LECs from collecting lymphatics147. Antibodies to LYVE1 have also been used to image lymphangiogenesis in vivo in mice by positron emission tomography135. Podoplanin is a mucin-type transmembrane glycoprotein that seems to be expressed on LECs of all lymphatic vessel subtypes146, 147. In human tissues, podoplanin can be detected with the widely available D2-40 monoclonal antibody148. Studies of lymphatic development and function will undoubtedly identify more lymphatic-specific markers that might be used to identify these vessels in cancer and to identify specific subtypes of tumour lymphatics.

Figure 1: Structure of the lymphatic vasculature and of lymphatic endothelial cells from initial lymphatics.
Structure of the lymphatic vasculature and of lymphatic endothelial cells from initial lymphatics.

The hierarchy of lymphatic vessel subtypes is shown in part a, with the characteristics of the subtypes indicated. The association between lymphatic endothelial cells (LECs) in initial lymphatics is shown in the two pull-out boxes, with the contact regions between LECs in the vessels being indicated by the brown lines in the first pull-out box. Fluid is thought to enter (as shown by the arrows in the second pull-out box) via the tips of the flaps of LECs, without compromising the integrity of the intercellular junctions on the sides of the flaps13. The structure of LECs from initial lymphatics is shown in part b, illustrating the entry of fluid and cells, which is favoured when the interstitial fluid pressure is high, causing stretching of the extracellular matrix. The structure of a blood vascular capillary is shown for comparison. The anchoring filaments that are associated with initial lymphatic vessels connect the abluminal membrane of LECs to surrounding elastic fibres — these filaments are short, but for visual clarity they are shown larger than to scale. Anchoring filaments are involved in controlling the entry of cells and interstitial fluid into the initial lymphatics. It should be noted that cells can cross lymphatic endothelial layers via a transcellular route, as well as via the paracellular route that is shown here. BEC, blood vascular endothelial cell; BM, basement membrane; SMC, smooth muscle cell.

Figure 2: Remodelling of lymphatic vessels in cancer and its contribution to metastasis.
Remodelling of lymphatic vessels in cancer and its contribution to metastasis.

Lymphatic vessels that are evident in adults arise during embryonic development. In cancer, these pre-existing lymphatics undergo various types of remodelling, including lymphangiogenesis (resulting in the generation of new lymphatics) and lymphatic enlargement. Lymphatic enlargement does not generate new vessels and can involve proliferative or non-proliferative mechanisms. Lymphangiogenic growth factors, which are derived from tumour cells or immune cells in the tumour microenvironment, promote tumour lymphangiogenesis and/or lymphatic enlargement in and around the primary tumour. These proteins also promote lymph node lymphangiogenesis, enlargement of tumour-draining collecting lymphatics (both proximal and distal to sentinel lymph nodes (SLNs; identified using a tracer)) and alterations to lymphatic smooth muscle cells (LSMCs) around collecting lymphatics6, 7, 8. These processes are thought to facilitate metastasis. The mechanism of transit of tumour cells from lymph nodes to distant organs is currently speculative.

Lymphangiogenesis

Lymphangiogenesis requires the coordination of complex cellular events, including proliferation, sprouting, migration and tube formation; these events might be similar to processes that occur in angiogenesis. The survival, proliferation and migration of LECs that are central to lymphangiogenesis depend on vascular endothelial growth factor receptor 2 (VEGFR2) and VEGFR3 signalling that is driven by VEGFC198 or VEGFD199, which can stimulate protein kinase C-dependent activation of the ERK1 or ERK2 signalling cascade and phosphorylation of AKT18, 19. In addition, the VEGFR3 co-receptor neuropilin 2 (NRP2) modulates the signalling pathways that are activated in response to VEGFC and VEGFD20, 21. These signalling events are somewhat analogous to the molecular regulation of angiogenesis by VEGFA signalling via VEGFR2 and NRP1 (Ref. 22). Furthermore, the cellular events that characterize angiogenesis, such as tip and stalk cell differentiation that is required for vessel sprouting23, 24, might be similar in lymphangiogenesis but the molecular mechanisms, which involve molecules such as VEGFC, NRP2, Notch 1 and Delta-like 4 (DLL4), are only beginning to emerge25. Blood vascular endothelial cells (BECs) and LECs share common molecular and functional traits, which have made it difficult to distinguish between blood vessels and lymphatics, especially in the tumour microenvironment, in which angiogenesis, lymphangiogenesis and remodelling of both vascular networks can occur. Nevertheless, LECs differ from BECs in their molecular and physiological behaviour26, 27, 28, 29. This is exemplified by their distinct gene expression and functional characteristics30, as well as by their independent specification and development in the embryo15, 16, 17, 31. Thus, although there are parallels between LECs and BECs, there are clear distinctions between these cells.

Various other signalling mechanisms that are important for lymphatic development have been defined (reviewed in Ref. 32). For example, a new lymphangiogenic signalling component, collagen and calcium binding EGF domain-containing protein 1 (CCBE1), was recently identified in a zebrafish mutagenesis screen. Mutations in ccbe1 led to the absence of the thoracic duct and dorsal lymphatics, whereas the blood vasculature was normal33. Although CCBE1 functions in the same developmental steps as VEGFC to promote lymphatic sprouting, the biochemical function of CCBE1 in cancer is unknown.

Primary tumour lymphangiogenesis

Lymphatic remodelling at the primary tumour. LECs have an active role in the interactions of tumour cells with lymphatic vessels34. The proliferation and migration of LECs contributes to the sprouting of lymphatic vessels, and LEC proliferation can be a feature of lymphatic enlargement; these forms of lymphatic remodelling have been shown to occur in various primary human cancers, of which melanoma is one example35. Most research on lymphatic vessels at the primary tumour has focused on the capacity of lymphatics to facilitate the entry and transport of tumour cells; the influence of lymphatic location (intratumoural versus peritumoural); and the enlargement or collapse of lymphatics during metastasis36, 37.

In the past, it was thought that there were almost no lymphatic vessels within tumours36. However, tumour-derived lymphangiogenic growth factors such as VEGFC and VEGFD can drive the formation of intratumoural lymphatic vessels, although the functional importance of these vessels remains questionable because they mostly seem to be collapsed37, 38. In fact, various studies in mouse models of cancer (Box 2) indicated that neither intratumoural lymphangiogenesis nor intratumoural lymphatics were required for lymph node metastasis37, 39. In general, intratumoural lymphatics have not been considered to be crucial for increasing the metastasis of tumour cells from primary tumours to lymph nodes, although it is nevertheless possible that they could provide an interface for lymphatic vessel invasion and metastasis40, 41, 42.

Box 2: Mouse models of metastasis

The experimental model of cancer that is used to monitor tumour lymphangiogenesis and metastasis can substantially influence the outcome and interpretation of disease severity. Xenograft models are widely used but can lack the natural physiological surroundings of the tumour and necessitate the use of immunocompromised animals. Nevertheless, xenograft tumours can recruit blood and lymphatic vessels, and they can subsequently metastasize to lymph nodes and distant organs.

Orthotopic xenografts are implanted into the correct organ or location and consequently might better represent the human disease. Furthermore, syngeneic models use mouse tumour cells and eliminate any differences in signalling pathways between human and mouse cells.

Tail-vein injection models are commonly used for assessing metastatic potential and the ability of cells to establish tumours in distant organs. However, these models do not involve some of the multiple steps of metastasis that occur in human disease, and they overlook the role of lymphatic metastasis.

Transgenic models that involve defined changes to the mouse genome facilitate the development of spontaneous tumours, and these models are used because they might better represent human disease. Transgenic models offer the flexibility of conditional oncogene expression, tissue-specific promoters and gene mutations that are directly relevant to human disease.

Studies that involve human tissues and animal models indicate that peritumoural lymphatics can undergo various degrees of morphological change, such as enlargement, as a result of the effects of tumour-secreted VEGFC and VEGFD36, 37, 43, 44. Peritumoural lymphatic vessels have been reported to enlarge in a manner that might involve proliferation of LECs (that is, hyperplasia), and the greater resulting surface area of potential contact between lymphatic vessels and tumour cells is thought to facilitate the entry of tumour cells into lymphatics and to be of functional importance for the metastatic spread of cancer36. Overall, the remodelling of peritumoural lymphatics in cancer seems to be a somewhat chaotic process42.

Molecular mechanisms of tumour-associated lymphangiogenesis. The VEGFC–VEGFR3 and VEGFD–VEGFR3 axis is considered to be a major driver of tumour lymphangiogenesis16, whereas the roles of other pathways in this process are less well defined. VEGFC and VEGFD, which are activated by proteolytic processing45, 46, 47, 48, 49, 50, both promote tumour-associated lymphangiogenesis and lymphatic metastasis in animal tumour models2, 3, 4. They also induce enlargement of collecting lymphatic vessels that are proximal and distal to SLNs; this enlargement can involve proliferation of LECs, non-proliferative mechanisms and alterations to lymphatic VSMCs57, 44. VEGFC and VEGFD are often expressed in primary human tumours or their associated stroma; they are secreted by tumour cells, immune cells and tumour-associated fibroblasts15, 51, 52, 53 (Fig. 3). The VEGFC and VEGFD signalling pathway has been targeted in experimental tumour models using a soluble form of VEGFR3 to sequester VEGFC and VEGFD, or using neutralizing VEGFR3-specific monoclonal antibodies (mAbs), which resulted in a restriction of tumour lymphangiogenesis and lymph node metastasis44, 54, 55, 56, 57, 58, 59. In addition, blocking NRP2 using a neutralizing NRP2-specific mAb prevented LEC migration (but not proliferation) both in vitro and in vivo, and it reduced the incidence of lymph node metastasis in experimental models25, 60.

Figure 3: Mechanisms of tumour lymphangiogenesis and interactions between tumour cells and lymphatics.
Mechanisms of tumour lymphangiogenesis and interactions between tumour cells and lymphatics.

Molecules that are reported to modulate tumour lymphangiogenesis are shown, with soluble ligands presented outside the cell, cognate receptors at the cell surface and transcription factors in the nucleus. Vascular endothelial growth factor C (VEGFC) and VEGFD refer to the proteolytically processed, biologically active forms of these proteins. Most of the ligands shown have been reported to promote lymphangiogenesis, whereas transforming growth factor-β (TGFβ) is considered to be an inhibitor of lymphangiogenesis. There are other molecules that are known to participate in lymphatic development in the embryo, such as collagen and calcium binding EGF domain-containing protein 1 (CCBE1; not shown), for which a role in tumour lymphangiogenesis has not been shown. The interaction of tumour cells with lymphatic vessels can be promoted by interstitial fluid flow (which partly results from lymphatic drainage) via autologous chemotaxis98 that involves chemokines, such as CC-chemokine ligand 21 (CCL21), and their receptors (CCR7 in the case of CCL21), which are expressed by tumour cells. Expression of CCL21 on lymphatic endothelial cells (LECs) can promote the entry of tumour cells into lymphatics via a CCR7-dependent mechanism. Production of lymphangiogenic growth factors, such as VEGFC and VEGFD, can drive the formation of new lymphatics and lymphatic enlargement in the vicinity of a tumour, which increases the surface area for the interaction of tumour cells with lymphatics. VEGFC can also promote tumour cell invasiveness in an autocrine manner, and it can upregulate the production of CCL21 on lymphatic vessels100. 15-PGDH, 15-hydroxyprostaglandin dehydrogenase; ANGPT2, angiopoietin 2; COUPTF2, COUP transcription factor 2; COX2, cyclooxygenase 2; CSF1, colony-stimulating factor 1; EGF, epidermal growth factor; EGFR, EGF receptor; EPO, erythropoietin; EPOR, EPO receptor; FGF, fibroblast growth factor; FGFR, FGF receptor; FOXC2, forkhead box protein C2; PDGF-BB, platelet-derived growth factor BB; PDGFR, PDGF receptor; PROX1, prospero homeobox protein 1; RAMP2, receptor activity-modifying protein 2; S1P, sphingosine-1-phosphate; TGFβR, TGFβ receptor; VEGFR, VEGF receptor.

Various signalling systems modulate the VEGFC–VEGFR3 and VEGFD–VEGFR3 axis, thereby influencing tumour lymphangiogenesis. For example, WNT1 can suppress the expression of VEGFC in melanoma cells, which leads to reduced lymphangiogenesis and delayed lymph node metastasis in mouse models of melanoma61. Prostaglandins can promote lymphangiogenesis in cancer via VEGFC62. The enzyme that is involved in the synthesis of prostaglandins, cyclooxygenase 2 (COX2), and the prostaglandin receptors EP2, EP3 and EP4, which are expressed by tumour cells and immune cells, modulate the expression of VEGFC in the tumour microenvironment, and this leads to increased tumour lymphangiogenesis and metastasis in vivo62, 63, 64, 65, 66, 67. A further link between prostaglandins and tumour lymphangiogenesis was identified when VEGFD was shown to modulate the expression of 15-hydroxyprostaglandin dehydrogenase (15-PGDH), the enzyme that breaks down prostaglandin in endothelial cells, resulting in prolonged exposure of LECs in collecting lymphatics to prostaglandins and enlargement of these vessels, which was associated with an increased rate of tumour cell dissemination6. The effect of prostaglandins on lymphangiogenesis is not specific to cancer: they can also promote lymphangiogenesis in the setting of inflammation66.

Other growth factors and signalling pathways are also involved in lymphangiogenesis (Fig. 3). VEGFA promoted lymphangiogenesis and lymph node metastasis both in a mouse tumour xenograft model in which mouse fibrosarcoma cells were implanted into syngeneic mice68, and in transgenic mice that overexpressed VEGFA in skin and were subjected to a chemical regimen that induces skin carcinogenesis69. However, these effects of VEGFA were not observed in a subcutaneous tumour xenograft model in immunocompromised mice2. The disparity between these observations could indicate that the effect of VEGFA on lymphatic vessels in cancer depends on the level and location of VEGFA expression, the abundance of VEGFRs on nearby LECs or other factors. Angiopoietins (ANGPTs) can promote peritumoural lymphangiogenesis in experimental mouse models70, and an ANGPT2-blocking antibody reduced tumour lymphangiogenesis, as well as lymph node and lung metastasis in a mouse tumour xenograft model71. Fibroblast growth factor 2 (FGF2) can promote lymphangiogenesis and can synergize with VEGFC signalling to induce robust lymphatic vessels and tumour metastasis72. Furthermore, platelet-derived growth factor B (PDGF-B; which can bind as a homodimer (PDGF-BB) to its cognate receptors) induced tumour lymphangiogenesis and promoted lymph node metastasis in mice73, and this could not be prevented using a VEGFR3-targeted antibody73. The expression of PDGF receptors (PDGFRs) on lymphatic vessels was shown in this study73, which suggests that PDGF-BB might directly induce proliferation or differentiation of LECs. Additional evidence of a role for PDGF-BB in lymphangiogenesis comes from a study in which inhibition of hypoxia-inducible factor 1α (HIF1α) in a mouse model of breast cancer reduced PDGF-BB expression and restricted lymph node metastasis74. In a recent study, Yoo and co-workers75 used models of gastric cancer to show that the sonic hedgehog pathway can promote lymphangiogenesis and metastasis. Likewise, erythropoietin and adrenomedullin were reported to promote tumour lymphangiogenesis and metastasis76, 77, and the biologically active lipid sphingosine-1-phosphate has been implicated as a tumour-derived factor that stimulates both angiogenesis and lymphangiogenesis78. In addition, epidermal growth factor (EGF) has been reported to facilitate lymph node metastasis in melanoma by influencing tumour lymphangiogenesis79. Interestingly, in an orthotopic mouse model of head and neck squamous cell carcinoma, inhibition of mTOR decreased lymphangiogenesis in the primary tumour and prevented the dissemination of tumour cells to cervical lymph nodes80. In contrast to the lymphangiogenic effects of many of the molecules that are discussed above, transforming growth factor-β (TGFβ) has been reported to be a negative regulator of lymphangiogenesis in cancer: inhibition of endogenous TGFβ signalling in mouse xenograft models of pancreatic adenocarcinoma induced lymphangiogenesis in the presence of VEGFC81.

Lymphatic vessel growth requires interaction with the ECM, which provides structural support for the vessels and modulates lymphangiogenic signalling. LEC adhesion to the ECM protein fibronectin is mediated by α4β1 integrin, which is expressed on LECs. Genetic loss of α4β1 integrin blocked tumour-induced lymphangiogenesis, as well as metastasis to lymph nodes, in experimental tumour models82. In addition, antagonists of α4β1 integrin suppressed lymphangiogenesis and tumour metastasis in vivo82. The tetraspanin CD9 was found to be highly expressed on LECs and is required for signalling crosstalk between integrins and VEGFRs. The importance of CD9 during tumour lymphangiogenesis was shown by the genetic ablation of CD9, which led to decreased lymphangiogenesis and lymph node metastasis83.

Genome-wide functional screening approaches that involve embryos or LECs in vitro might provide an opportunity to identify other functionally important signalling pathways in lymphangiogenesis and to identify novel regulators of tumour lymphangiogenesis. Such approaches could also be used to globally compare signalling for lymphangiogenesis and angiogenesis, and they might help us to understand the differences in gene expression that occur in LECs isolated under different normal and pathological conditions. Compared to LECs from normal tissues, LECs that are isolated from metastatic tumours in mice show alterations in the expression of molecules that have a role in maintaining endothelial cell junctions, the sub-endothelial matrix and vessel growth and remodelling6, 84. Interestingly, the molecular profile of tumour LECs is also distinct from that of LECs that are isolated from an inflammatory microenvironment and that of mitogen-activated LECs84. However, the underlying molecular mechanisms that drive altered gene expression in tumour LECs are not well understood.

Chemokines that drive metastasis. Lymphatic vessels function as conduits for cells of the immune system and thereby provide a route for antigen-presenting cells to transit from peripheral tissues to lymph nodes and other secondary lymphoid sites. Chemokines facilitate these cellular movements. For example, LECs can facilitate the entry of dendritic cells into lymphatic vessels by expressing CC-chemokine ligand 21 (CCL21), which is thought to remain mostly associated with the surface of LECs and can interact with its receptor CCR7, which is located on dendritic cells85. Once in the lymphatic system, dendritic cells can be carried with afferent lymph into draining lymph nodes. Recent studies have shown that tumour cells can also enter or be attracted to lymphatics through chemokine-dependent mechanisms that have been implicated in cancer metastasis86 (Fig. 3). Various tumour models have shown that cancer cells that express certain chemokine receptors undergo increased rates of metastasis to lymph nodes that express the appropriate chemokine87, 88, 89, 90. Clinicopathological data also support the idea that chemokines and their receptors are involved in lymphatic metastasis; for example, the expression of CCR7 by tumour cells is associated with lymph node metastasis of gastric carcinoma, colorectal carcinoma and breast cancer91, 92, 93. Furthermore, expression of CXC-chemokine receptor 4 (CXCR4) by tumour cells significantly correlated with lymphatic metastasis and distant spread in hepatocellular carcinoma94, and an association with lymph node metastasis in oral squamous cell carcinoma95 and breast cancer96 has also been reported. CXCR4 is also expressed by invasive Paget cells in extramammary Paget's disease97. From a mechanistic perspective, the idea that CXCR4 is involved in the spread of tumour cells is supported by the observation that its ligand, CXCL12, which can be expressed by tumour-associated lymphatic vessels97, promotes the invasiveness of various types of tumour cells94, 95.

An elegant mechanism by which chemokines and their receptors can promote tumour cell homing to lymphatics was proposed based on the effect of interstitial fluid flow resulting from lymphatic drainage — this mechanism is known as autologous chemotaxis98. In model systems, tumour cells of various types were shown to secrete CCL21 and CCL19. The interstitial flow of fluid drives the formation of autologous gradients of these molecules. Given that the tumour cells also express a cognate CCL21 and CCL19 receptor, CCR7, the tumour cells can migrate along the chemokine gradient. Hence, this mechanism results in the movement of tumour cells in the direction of fluid flow: that is, towards lymphatic vessels98. Autologous chemotaxis has also been reported to have a role in the invasion of brain tissue by glioma cells99.

A link between VEGFC signalling and tumour cell invasion via chemotaxis is supported by the finding that tumour cell secretion of VEGFC, and the resulting increase in VEGFR3 signalling in LECs, led to increased CCL21 secretion by LECs100. In turn, this can increase the movement of CCR7-positive tumour cells towards LECs. A different mechanism by which chemokines might regulate metastasis involves the control of tumour cell entry into lymph nodes. The chemokine CCL1 was detected in lymph node lymphatic sinuses but not in peripheral lymphatics, and CCR8 (the CCL1 receptor) can be expressed by tumour cells101. Importantly, the blockade of CCR8 function in a mouse model restricted lymph node metastasis and caused arrest of tumour cells in collecting lymphatics at the junction with the subcapsular sinus of the lymph node. Hence, CCL1 and CCR8 could be involved in a checkpoint for the entry of tumour cells into lymph nodes101. Overall, there is an expanding body of data indicating that chemokines can promote the spread of cancer via the lymphatic vasculature through various mechanisms.

Remodelling beyond the primary tumour

Remodelling of collecting lymphatics. Research on the mechanisms that govern lymphogenous spread has predominantly focused on the effects of lymphangiogenic growth factors on initial and pre-collecting lymphatics in and around the primary tumour, with the collecting lymphatic vessels that drain the tumour being essentially ignored. The initial lymphatics in and around a tumour generally respond to lymphangiogenic growth factors by proliferation and/or sprouting, whereas collecting lymphatics respond to tumour-derived lymphangiogenic stimuli differently38, 43, 44, 102 (Fig. 2). Collecting lymphatics, which have traditionally been considered to be passive conduits during metastasis, can undergo substantial remodelling during the course of lymphogenous spread6, 7, 44. Observations from animal models of VEGFC-driven lymphogenous metastasis indicate that collecting lymphatics that drain the primary tumour might have an active role in metastasis. The presence of VEGFC promoted enlargement of collecting lymphatics, which was attributed to the proliferation of LECs, and this was associated with an increase of the total volumetric flow rate in the lymphatics43, 44, 102. Similarly, it has been shown that tumour-derived VEGFD promotes the enlargement of tumour-draining collecting lymphatics and that this was required for the dissemination of cancer cells to SLNs and distant organs6. However, the mechanism by which circumferential enlargement of the collecting vessels occurred was not through LEC proliferation but instead through prostaglandin-mediated responses6. There is also evidence for remodelling of collecting lymphatics distal to SLNs, and this remodelling might influence metastasis. Increased VEGFC-mediated lymph transport was associated with structural alterations to collecting lymphatics beyond SLNs7. The structural remodelling involved lymphatic VSMCs, and increased levels of post-SLN metastasis required the expression of NRP2 (Ref. 7).

Sentinel and regional lymph node metastatic niches. The expansion of the lymphatic network in the draining lymph nodes has been referred to as the 'lymphvascular niche' (Ref. 103). This is thought to be structurally similar to the 'vascular niche' that is necessary for the formation and maintenance of haemopoietic stem cells in the lymph nodes and the bone marrow103, 104. VEGFC and VEGFD that are produced at the primary tumour can modulate lymphatic vessels and blood vessels, including high endothelial venules (HEVs), in lymph nodes before the arrival of metastatic cells8, 69, 105, 200. The resulting increase in lymphatic vessel density in the lymph nodes contributes to the formation of the lymphvascular niche, and this might promote the transport of tumour cells to the SLN and serve as an indicator of future lymphogenous spread103. For example, it was recently shown that the lymphatic microenvironment at preferred metastatic sites, such as lymph nodes, guided the metastasis of distinct melanoma subsets that showed chemoresistance; this guidance was dependent on the CXCL12–CXCR4 chemotactic axis106. Furthermore, it has been speculated that the altered lymphatic microenvironment in lymph nodes might provide a location for the residence and survival of metastatic cancer cells, tumour cells that are resistant to chemotherapy or cancer stem cells; the increased survival of these cells might also be promoted by the CXCL12–CXCR4 axis103, 106. The lymphangiogenesis that creates the lymphvascular niche might also influence immune responses to tumour cells that eventually enter the lymph node, thereby potentially generating permissive conditions for the expansion of micrometastases. These processes might be similar to how vascular niches support the initial small foci of metastatic cells in distant organs107, 108.

Lymphatic remodelling and tumour immunity. Many studies of tumour lymphangiogenesis driven by VEGFC or VEGFD have been conducted in immunocompromised mice; hence, they have ignored the effects of lymphangiogenesis on tumour immunity. LECs share some characteristics with professional antigen-presenting cells (reviewed in Ref. 109). In addition, LECs in lymph nodes and in tissue lymphatics express major histocompatibility complex class II (MHCII) and MHCI molecules, and these LECs have been shown to function as antigen-presenting cells and to induce self-tolerance109, 110. In this context, it is interesting to note that VEGFC was reported to both protect melanoma cells from antitumour immunity and increase lymphangiogenesis around primary tumours and in draining lymph nodes111. Importantly, LECs in draining lymph nodes cross-presented tumour-associated antigen and promoted the apoptosis of tumour-specific CD8+ cells. Lymphatic presentation of antigen is a newly described and potentially important mechanism by which tumours evade host immune responses112, and it warrants further investigation in animal models and in a clinicopathological setting.

Tumour lymphangiogenesis in pathology

As discussed above, lymphangiogenesis and the enlargement of lymphatics can occur in and around human tumours, and this has been correlated with metastasis and shorter disease-free survival35 (Table 1). Furthermore, the expression of lymphangiogenic growth factors113 and the invasion of lymphatic vessels by tumour cells114 might be useful early indicators of whether metastasis to lymph nodes and distant organs is likely to occur (Table 1). It will therefore be important to determine whether the analysis of tumour lymphatics could be used before lymph node metastasis has occurred to stratify patients for therapy. In contrast to the status of tumour lymphatics, lymph node status (that is, the presence or absence of cancer cells) is already recognized as a significant and independent prognostic factor in many human cancers. It correlates with key clinical parameters, such as disease-free survival and overall survival, and it is often a more reliable parameter than tumour size or histological grade115, 116. In the past, large groups of lymph nodes were removed for pathological analysis; however, current techniques, such as SLN biopsy in melanoma117 and breast cancer118, can predict tumour spread with a considerable degree of accuracy, with minimal resection of lymph nodes.

Table 1: The relationship between tumour lymphatic parameters and patient outcome

The detection of circulating tumour cells or tumour-derived products (such as exosomes, nucleic acids and peptides) in the blood has become a focus for the development of diagnostic or prognostic approaches for cancer119, 120, 121. Similarly, it might be beneficial to screen lymph fluid for such tumour-derived products but this would require overcoming the technical hurdle of collecting lymph fluid from cancer patients.

A key challenge in cancer biology is to identify the specific pathways of dissemination that are taken by metastatic tumour cells to reach distant organs. Two contrasting models are used to explain the formation of distant organ metastases: a lymphatic-independent haematogenous model and a lymphatic-dependent sequential model122. According to the haematogenous model, blood vessels invade the tumour and are responsible for carrying tumour cells to distant organs. Moreover, the spread of tumour cells to regional lymph nodes is thought to reflect the aggression of the primary tumour, with no contribution of tumour cells in the lymph nodes to metastasis to distant organs. In the lymphatic-dependent (sequential) model, tumour cells spread to regional lymph nodes through lymphatics and thereafter enter the bloodstream, potentially through a remodelled vasculature in lymph nodes or into the subclavian vein via the thoracic duct, and they subsequently spread to distant organs8, 123, 124. One potential approach to clarify the relative importance of these two models would be to develop animal models of metastasis in which the tumour cells become labelled (using inducible promoters) only when they come into contact with molecules that are encountered in lymph nodes. The contribution of these labelled cells to distant organ metastasis could then be quantified.

Correlation of lymphangiogenesis and lymphatic remodelling with outcomes in human cancer. Studies of human malignant melanoma have shown that parameters of lymphangiogenesis and lymphatic remodelling are correlated with patient outcomes (Table 1). For example, clinical and histological analyses of closely matched cases of primary melanomas with early lymph node metastasis and primary non-metastatic melanomas showed that the incidence of intratumoural lymphatics was significantly higher in metastatic melanomas and correlated with poor disease-free survival35. Metastatic melanomas also had enlarged tumour-associated lymphatics, and lymphatic vessel area was significantly associated with poor disease-free and overall survival. These findings led to the proposal that tumour lymphangiogenesis might be a novel prognostic indicator for the risk of lymph node metastasis in cutaneous melanoma. Another study in cutaneous melanoma indicated that lymphatic invasion at the primary tumour is associated with metastasis to SLNs and distant metastasis125.

Similar findings have been reported for other tumour types (Table 1). For example, immunohistochemical staining of resected colorectal carcinomas for the lymphatic vessel marker D2-40 (also known as podoplanin) showed that a high density of lymphatic microvessels had prognostic significance, as it correlated with metastasis to regional lymph nodes and the liver126. Lymph node metastasis is a key prognostic factor for the survival of patients with breast cancer, and there are clinical data indicating that the spread of breast cancer cells to lymph nodes is facilitated by lymphangiogenesis, although this has been an area of controversy. Some studies in breast cancer have reported difficulties in detecting dividing LECs and other studies have reported reduced lymphatic vessel density. These findings suggest that pre-existing lymphatic vessels — rather than new vessels generated through lymphangiogenesis — are associated with metastasis (reviewed in Ref. 122). Interestingly, inflammatory breast cancer contains a significantly higher proportion of proliferating LECs than non-inflammatory breast cancer127. Hence, lymphangiogenesis might help to explain the propensity of inflammatory breast cancer to spread through the lymphatic vasculature.

In some human tumours, lymphangiogenesis is not present or detected, even though the spread of tumour cells to the lymph nodes can occur128, 129. An example is pancreatic ductal adenocarcinoma, for which early lymph node metastasis is common. A clinicopathological study showed that the average density of the intratumoural lymphatic vessel was decreased in this disease compared to the normal pancreas, and there was no overexpression of VEGFC or VEGFD in the primary tumours compared to normal pancreatic tissue129. It has been proposed that lymphatic metastasis predominantly occurs via pre-existing peritumoural lymphatics in this disease, and this is consistent with findings from animal models of prostate cancer39.

Mechanisms that lead to the enlargement and remodelling of collecting lymphatic vessels have been proposed to contribute to increased lymph flow, tumour cell dissemination, and lymph node metastasis and metastasis to distant organs6, 7, 44. However, the clinicopathological importance of the enlargement of collecting lymphatic vessels that are either proximal or distal to tumour-draining lymph nodes has yet to be extensively explored and might require the development of novel imaging approaches that allow easy and reliable monitoring of collecting lymphatics in cancer patients.

Correlation between lymphangiogenic growth factors and metastasis. Various studies have shown that the expression of lymphangiogenic growth factors and/or their receptors in primary tumours can be prognostic indicators in human cancer123, 130 (Table 1). For example, patients with high levels of VEGFC expression in gastric cancer had poorer prognoses than those with tumours that expressed low levels of this protein131, and VEGFC levels in primary tumours have been reported to correlate with lymph node metastasis in lung, oesophageal, prostate, thyroid and colorectal cancer (reviewed in Ref. 130). Likewise, the expression of VEGFD and VEGFR3 may be predictive of myometrial invasion and lymph node metastasis in endometrial carcinoma132, and VEGFD was reported to be an independent prognostic indicator of poor disease-free and overall survival in patients with colorectal cancer133 (Table 1).

Overall, recent clinicopathological findings indicate that there might be opportunities for improved approaches to prognostication based on monitoring tumour tissue samples for the expression of lymphangiogenic growth factors and their receptors, for the density of lymphatic vessels (intratumoural and/or peritumoural) and for lymphatic invasion (Fig. 4). Experimental studies using animal models have also indicated that it could be beneficial to monitor lymph nodes for lymphangiogenesis114 and to monitor tumour-draining collecting lymphatics for tumour-induced enlargement6, 7, 44. Importantly, imaging approaches are currently being developed that might allow these parameters to be monitored in vivo134, 135, 136.

Figure 4: Lymphangiogenesis and lymphatic remodelling in cancer — implications for diagnostics.
Lymphangiogenesis and lymphatic remodelling in cancer [mdash] implications for diagnostics.

Using breast cancer as an example, the anatomical features affected by lymphangiogenesis and lymphatic remodelling in cancer are shown in the boxes in bold; the boxes also show the changes that occur to these features in cancer and the actual or potential diagnostic approaches for monitoring these changes. ELISA, enzyme-linked immunosorbent assay; I-PET, immuno-positron emission tomography.

Targeting lymphatic vessels in cancer

The VEGFC–VEGFR3 and VEGFD–VEGFR3 axis is the most studied lymphangiogenic signalling pathway137. Many studies of tumour xenograft models in mice have shown that targeting this pathway restricts tumour lymphangiogenesis, lymphatic enlargement and lymph node metastasis2, 5, 15, 41, 44, 57, 58, 123. The finding that tumour lymphangiogenesis might be a useful prognostic indicator of lymph node metastasis in human melanoma35, 138 and other human cancers (discussed above) is consistent with the concept that targeting tumour lymphangiogenesis in the clinic might restrict metastasis to lymph nodes and potentially to distant organs. Importantly, targeting VEGFR3 can also restrict angiogenesis, given that this receptor is expressed on angiogenic sprouts and contributes to blood vessel formation23, 59, 139, 140. Targeting the VEGFC–VEGFR3 and VEGFD–VEGFR3 axis could have the benefit of restricting solid tumour growth (through anti-angiogenic effects) and lymph node metastasis (through anti-lymphangiogenic effects), although the degree to which metastases in lymph nodes contribute to metastasis to distant organs remains uncertain. A further potential benefit of restricting tumour cell migration to lymph nodes relates to immune function, given that tumour-draining lymph nodes might be able to promote immune tolerance to tumour antigens141.

Various inhibitors that target the VEGFC–VEGFR3 and VEGFD–VEGFR3 axis have been developed that could be useful in the clinic, including neutralizing mAbs to VEGFC, VEGFD, VEGFR3 and NRP2, as well as a soluble version of VEGFR3 that is capable of sequestering VEGFC and VEGFD (Table 2). As of 2013, the mAbs that target VEGFC and VEGFR3 have progressed to Phase I clinical trials in patients with advanced solid tumours (Table 2). Furthermore, several small-molecule protein kinase inhibitors (PKIs) have been developed that target various kinases, including VEGFR3. Some of these PKIs have been approved for the treatment of various cancers, although their effects on lymphangiogenesis may not have been considered during the approval process (Table 2). Although the treatment of cancer patients with both PKIs and cytotoxic chemotherapies could have synergistic effects, efforts to use these combinations have been limited because of dose-limiting toxicities. Other potentially useful reagents include antibodies and small molecules that target the ANGPT–ANGPT1 receptor (TIE2; also known as TEK) signalling axis71, 142 (Table 2). Some of these agents have been reported to restrict both tumour lymphangiogenesis and metastasis in animal models. An alternative approach could involve non-steroidal anti-inflammatory drugs (NSAIDs) that target COX enzymes, which are important for the enlargement of tumour-draining collecting lymphatics6 (Table 2).

Table 2: Potential targets and inhibitors for tumour lymphangiogenesis

Animal-based studies of anti-lymphangiogenic therapeutics for the treatment of cancer have not yet identified any particularly substantial acute side effects. However, it remains unclear as to when such drugs should be ideally given to cancer patients to restrict cancer metastasis. The treatment of most solid tumours involves surgery to remove the primary tumour for local disease control and to restrict metastasis. Patients who are thought to have a substantial risk of distant relapse are then offered adjuvant therapy143, which is typically cytotoxic chemotherapy. An anti-lymphangiogenic strategy could be used in conjunction with conventional chemotherapy regimens in this scenario. In addition, anti-lymphangiogenic agents could be used as part of pre-operative treatment regimens that are designed to reduce the size of the primary tumour to facilitate surgery. This approach would also allow a direct analysis of the effect of the anti-lymphangiogenic agent on the tumour lymphatics. If the data from these studies were to indicate a reduction in tumour lymphatic density, then a pre-operative approach for many cancer types might be beneficial, as some tumours might seed metastasis from an early stage or a small size144. In theory, these anti-lymphangiogenic drugs could also be used, in combination with other systemic agents, for the treatment of patients with advanced, non-resectable cancers. In this scenario, the inclusion of anti-lymphangiogenic agents would aim to reduce the incidence of local or distant recurrence9.

More work on tissue-banked specimens from human cancers is required to identify the best candidate cancers for clinical trials on the basis of the detection of lymphangiogenesis in or adjacent to the primary tumour and invasion of lymphatics by tumour cells. These studies could be augmented by new imaging approaches, such as immuno-positron emission tomography (I-PET) with lymphatic-specific antibodies, to identify tumour types for which lymph node lymphangiogenesis and enlargement of tumour-draining collecting lymphatics are prevalent10 (Fig. 4). Images that are obtained using such technology might be useful for patient selection, and I-PET images might also be useful for monitoring disease progression during treatment with anti-lymphangiogenic agents. Reliable assays for increased concentrations of lymphangiogenic growth factors in the blood might also be required. Ultimately, detailed clinical trials that involve biological and clinical end points will be required to test the efficacy of anti-lymphangiogenic approaches for cancer therapy.

Conclusions and perspectives

Questions remain as to how lymphatic vessels contribute to the metastatic spread of cancer. First, the molecular mechanisms that govern the entry of tumour cells into new or remodelled lymphatics — be they intratumoural or peritumoural — have not been fully characterized. Second, it is not clear whether tumour lymphangiogenesis and/or the remodelling of lymphatics are absolutely required for the spread of tumour cells to lymph nodes or distant organs. Third, the degree to which lymph node metastases directly contribute to distant organ metastasis needs to be defined, as do the mechanisms through which this may occur. Animal models in which tumour cells become inducibly labelled during their passage through lymph nodes could be developed to determine the contribution of lymph node spread to distant organ metastasis. Some of these questions might also be addressed by the selective and inducible disruption of specific lymphatic vessel subtypes in mouse models that is directed through lymphatic-specific transcriptional promoters. This could help to determine the contribution of different subtypes of lymphatics, and the contribution of the blood vascular system, to lymph node and distant organ metastasis. In addition, mice with gene mutations that drive lymphatic-specific or related phenotypes might help to further characterize the role of the lymphatic vasculature in cancer metastasis.

The extensive characterization of human cancer genomes that is currently underway will provide an opportunity to identify which gene mutations in cancer cells lead to patterns of gene expression that favour metastasis through the lymphatics (such as high expression of the VEGFC and/or VEGFD genes). This information could be considered in combination with clinicopathological data about lymphatic vessels in and around the tumour. The progress of current clinical trial programmes using agents that target lymphangiogenic signalling pathways might also shed light on the role of the lymphatics in metastasis, although clinical and biological end points for the trials will need to be appropriately designed to assess effects on metastasis (in addition to monitoring effects on the primary tumour). The capacity to use new molecular technologies, such as novel imaging approaches and whole-genome functional screens with LECs, will help to identify the molecular and cellular characteristics of specialized lymphatics in different organs. This should facilitate the discovery of novel diagnostic and therapeutic targets, and it should enhance our understanding of the contribution of the lymphatic system to tumour progression in cancer patients.

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Acknowledgements

S.A.S., T.K. and M.G.A. are supported by Project Grants, a Program Grant and Research Fellowships from the National Health and Medical Research Council (NHMRC) of Australia, and by funds from the Operational Infrastructure Support Program that is provided by the Victorian Government, Australia. R.S. has been supported by the Raelene Boyle Sporting Chance Cancer Foundation and the Royal Australasian College of Surgeons (RACS) Foundation Scholarship, as well as the RACS Surgeon Scientist Program. S.P.W. has been supported by an Australian National Breast Cancer Foundation Doctoral Research Fellowship. The authors thank M. Macheda for proofreading.

Author information

Affiliations

  1. Tumour Angiogenesis Program, Peter MacCallum Cancer Centre, East Melbourne, Victoria 3002, Australia.

    • Steven A. Stacker,
    • Steven P. Williams,
    • Tara Karnezis,
    • Ramin Shayan &
    • Marc G. Achen
  2. Sir Peter MacCallum Department of Oncology, University of Melbourne, Victoria 3010, Australia.

    • Steven A. Stacker,
    • Tara Karnezis,
    • Stephen B. Fox &
    • Marc G. Achen
  3. Department of Surgery, Royal Melbourne Hospital, University of Melbourne, Parkville, Victoria 3050, Australia.

    • Steven A. Stacker,
    • Ramin Shayan &
    • Marc G. Achen
  4. Department of Surgery, St. Vincent's Hospital, University of Melbourne, Fitzroy, Victoria 3065, Australia.

    • Ramin Shayan
  5. O'Brien Institute, Australian Catholic University, Fitzroy, Victoria 3065, Australia.

    • Ramin Shayan
  6. Department of Pathology, Peter MacCallum Cancer Centre, East Melbourne, Victoria 3002, Australia.

    • Stephen B. Fox

Competing interests statement

S.A.S. and M.G.A. have both been consultants for Vegenics Limited, South Yarra, Victoria, Australia, in the area of developing inhibitors of angiogenesis and lymphangiogenesis in human diseases, including cancer. This consultancy ended on 31 December 2012. S.A.S., R.S. and M.G.A. are shareholders in Circadian Technologies (owner of Vegenics), South Yarra, Victoria, Australia, which is a company that develops anticancer therapeutics, and in Ark Therapeutics Group Plc, London, UK, which has been developing therapeutic approaches based on the vascular endothelial growth factor family. S.A.S., S.B.F. and M.G.A. are holders of an Australian National Health and Medical Research Council (NHMRC) Program Grant. S.A.S. and M.G.A. are holders of NHMRC Senior Research Fellowships in this area. S.A.S., S.B.F., T.K. and M.G.A. are holders of NHMRC Project Grants in this area. S.A.S. and M.G.A. are holders of numerous patents in this area. S.P.W. declares no competing interests.

Corresponding authors

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Author details

  • Steven A. Stacker

    Steven A. Stacker is a joint-head of the Tumour Angiogenesis Program at the Peter MacCallum Cancer Centre, Melbourne, Victoria, Australia, and has a long-standing interest in basic and translational research into the role of blood vessels and lymphatics in cancer. His work on growth factors and receptors has led to genome-wide approaches to map complex signalling pathways in the endothelium. Steven A. Stacker's homepage.

  • Steven P. Williams

    Steven P. Williams completed his Ph.D. in the Tumour Angiogenesis Program at the Peter MacCallum Cancer Centre, Melbourne, Victoria, Australia, and the University of Melbourne, Victoria, Australia. His main interest lies in using whole-genome approaches to understand the signalling pathways that regulate endothelial cell function.

  • Tara Karnezis

    Tara Karnezis is a senior research fellow in the Tumour Angiogenesis Program at the Peter MacCallum Cancer Centre, Melbourne, Victoria, Australia. She has a keen interest in lymphatic endothelial cell biology — in particular, how lymphatic vessels are remodelled during various pathologies — with an emphasis on metastatic disease.

  • Ramin Shayan

    Ramin Shayan is a plastic surgeon and an active researcher. His Ph.D. in the Tumour Angiogenesis Program, Peter MacCallum Cancer Centre, Melbourne, Victoria, Australia, and the University of Melbourne, Victoria, Australia, was focused on characterizing lymphatic vessel subtypes and their role in cancer. His long-term goal is to use his research into blood vessels and lymphatics to improve outcomes for his patients.

  • Stephen B. Fox

    Stephen B. Fox is Director of Pathology at the Peter MacCallum Cancer Centre, Melbourne, Victoria, Australia, and a professorial fellow at the University of Melbourne, Victoria, Australia. He is a clinician-researcher investigating predictive markers of response to therapies in several tumour types using protein and DNA-based assays. His goal is to develop novel assays for cancer diagnostics.

  • Marc G. Achen

    Marc G. Achen is a joint-head of the Tumour Angiogenesis Program at the Peter MacCallum Cancer Centre, Melbourne, Victoria, Australia. His research is focused on the molecular and biochemical mechanisms that control angiogenesis and lymphangiogenesis in cancer and other pathologies. He is also involved in translational research that aims to modulate angiogenesis or lymphangiogenesis in cancer and other diseases. Marc G. Achen's homepage.

Glossary

Lymphangiogenesis

The formation of new lymphatic vessels from pre-existing lymphatics.

Lymphatic enlargement

The enlargement of lymphatics, which can occur via proliferation of lymphatic endothelial cells (that is, hyperplasia) or other non-proliferative mechanisms.

Lymphatic hyperplasia

The enlargement of lymphatics by proliferation of lymphatic endothelial cells, which may or may not be accompanied by sprouting.

Initial lymphatics

Small blind-ended lymphatics in the tissue periphery that are adapted for the uptake of fluid and cells.

Collecting lymphatics

Large lymphatics that are characterized by a continuous smooth muscle cell coating and are adapted for the transport of lymph and associated cells.

Lymphatic remodelling in cancer

Alteration to the structure and morphology of lymphatic vessels that are associated with cancer, including lymphangiogenesis and lymphatic enlargement.

Lymphatic invasion

The entry of tumour cells into lymphatics, which is identified clinicopathologically by the detection of tumour cells or tumour cell emboli within lymphatics.

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