lymphangiogenesis and cancer metastasis - CiteSeerX

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LYMPHANGIOGENESIS AND CANCER METASTASIS Steven A. Stacker*§, Marc G. Achen*§, Lotta Jussila‡, Megan E. Baldwin* and Kari Alitalo‡ Lymphatic vessels are important for the spread of solid tumours, but the mechanisms that underlie lymphatic spread and the role of lymphangiogenesis (the growth of lymphatics) in tumour metastasis has been less clear. This article reviews recent experimental and clinico-pathological data indicating that growth factors that stimulate lymphangiogenesis in tumours are associated with an enhanced metastatic process. M E TA S TA S I S LYMPHATIC VESSELS

The system of thin-walled, lowpressure vessels that collects fluid, proteins and cells that are released by the blood vessels into the interstitial spaces of tissues. METASTASIS

The spread of malignant cells from the original site of the tumour. Spread occurs to such sites as the draining lymph nodes or to distant sites — typically the lung, liver or bone marrow in the case of solid tumours (for example, carcinomas — tumours of epithelial origin).

*Ludwig Institute for Cancer Research, Post Office Box 2008, Royal Melbourne Hospital, Victoria 3050, Australia. ‡ Molecular/Cancer Biology Laboratory and Ludwig Institute for Cancer Research, The Haartman Institute and Biomedicum Helsinki, P.O.Box 63 (Haartmaninkatu 8), 00014 University of Helsinki, Finland. §Contributed equally to the review. Correspondence to K. A. e-mail: [email protected] doi:10.1038/nrc863

Millions of people die every year from the metastatic spread of cancer that occurs via the blood vessels (haematogenous spread), LYMPHATIC VESSELS (lymphogenous spread) or directly into tissues and body cavities. Although the biochemical mechanisms are not well understood, it is thought that the metastatic spread of a tumour is not a random process. Distinct patterns of 1 METASTASIS can be discerned in different tumour types . A common pattern for carcinomas is that regional LYMPH NODES are the first sites to develop metastases, either draining via pre-existing afferent lymphatic vessels and/or via newly formed lymphatic capillaries. This pattern of metastasis is central to the usefulness of sentinel lymphadenectomy as a surgical technique2,3. However, not all tumours initially metastasize to the regional lymph nodes. The mechanisms that determine whether regional lymph nodes or other sites first develop metastases remain poorly understood. In fact, most disseminated tumour cells have a limited lifespan and only a few develop into detectable micrometastases3,4. Nevertheless, the ability to identify occult tumour cells, as well as prevent their growth and spread, would be of clinical significance. ANGIOGENESIS and lymphangiogenesis provide new vessels that malignant cells can use to escape the confines of the primary tumour. Nevertheless, existing vessels already provide a mechanism of escape, and therefore the relative importance of pre-existing versus new vessels is still to be determined5. Studies using various inhibitors and other approaches have shown that angiogenesis is important for solid tumour growth6–10 and, presumably, also in haematogenous metastasis11. However, little is known

about the role of tumour lymphangiogenesis in metastasis and whether this process is important for spread via the lymphatics12,13. Much of the difficulty in analysing lymphatics is due to the lack of lymphatic-specific markers that could be used to discriminate between lymphatics and blood vessels14. However, recent experimental and clinical data that employ lymphangiogenic factors and novel lymphatic markers indicate that lymphangiogenic factors influence the spread of tumours. Molecular mediators of lymphangiogenesis

The discovery of molecules that promote lymphatic vessel growth and allow the identification of lymphatics has brought a new awareness of the process of lymphangiogenesis15–21. The first lymphangiogenic growth factor discovered — vascular endothelial growth factor-C (VEGFC) (FIG. 1) — has a mature form that consists of the VEGF homology domain (VHD), which contains receptor-binding sites and is 30% identical in amino-acid sequence to VEGF165 (REFS 22,23). VEGFC is synthesized as a preproprotein, and several forms are generated, by means of stepwise proteolytic processing, that have sequentially increased affinity for VEGFR2 and VEGFR3 (REF. 24) — cell-surface-receptor tyrosine kinases that are predominantly expressed on bloodvascular and lymphatic ENDOTHELIA, respectively25 (FIG. 2). Like VEGF, VEGFC stimulates the migration of endothelial cells and induces vascular permeability and endothelial-cell proliferation, but at higher concentrations than are required for VEGF22,24. These signals for endothelial cells are probably mediated by means of

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© 2002 Nature Publishing Group

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Growth factors VEGFC (full length) N

Sig

N-Pro

VHD

C-Pro

C

VHD

C-Pro

C

VEGFD (full length) N

Sig

N-Pro

VEGF N

Sig

VHD

Hep

C

Ig5

Receptors VEGFR2 N

Sig

Ig1

Ig2

Ig3

Ig4

Ig1

Ig2

Ig3

Ig4 Ig S

Ig6

Ig7

TM

TK1

KI

TK2

CT

C

VEGFR3 N

Sig

S 5

Ig6

Ig7

TM

TK1

KI

TK2

CT C

Figure 1 | The protein structure of growth factors and receptors associated with lymphangiogenesis. Two members of the vascular endothelial growth factor (VEGF) family (VEGFC and VEGFD) are implicated in lymphangiogenesis — VEGF is shown for comparison. These are secreted homodimeric polypeptides that contain a hydrophobic amino-terminal signal sequence for protein secretion (Sig). All VEGF family members contain a central region that is termed the VEGF homology domain (VHD) and contains a cysteine-knot motif, a structural feature of each subunit that consists of three intra-subunit disulphide bonds forming the shape of a knot. A subfamily of these growth factors contains VEGFC and VEGFD, defined by their ability to bind VEGFR3 and the presence of amino- and carboxy-terminal propeptides (N-Pro and C-Pro). The receptors for VEGFC and VEGFD are VEGFR2 and VEGFR3, both of which can be expressed on lymphatic endothelial cells. These receptors are closely related in structure, consisting of an extracellular domain with seven immunoglobulin-like domains (Ig), a transmembrane domain (TM), a split tyrosine kinase domain (TK) with a kinase insert sequence (KI) and a cytoplasmic tail (CT) at the carboxyl terminus. The fifth Ig-like domain of VEGFR3 is proteolytically processed and the subunits are held together by a disulphide bond (-S-S-). Recent studies have revealed that signalling by VEGFR3 alone is sufficient to induce lymphangiogenesis20. Some forms of the angiogenic factor VEGF contain a carboxy-terminal heparin-binding domain (Hep). VEGF binds VEGFR1 (not shown here) and VEGFR2, but not VEGFR3.

VEGFR2 in blood-vascular-endothelial cells and generally by VEGFR3 (also known as FLT4) in lymphatic endothelial cells, although lymphatic endothelium

also expresses VEGFR2, which might contribute to signalling for mitogenesis. Unlike VEGF, the expression of VEGFC does not seem to be regulated by hypoxia26, but is increased in response to proinflammatory cytokines, indicating a role in inflammatory responses 27. The pattern of Vegfc expression in embryos indicates that it has a role in the development of the lymphatic vessels, since a paracrine expression pattern is seen between Vegfc and Vegfr3 at sites in which the first lymphatic sprouts occur28. In addition, deletion of the Vegfc gene results in embryonic lethality because of failure of lymphaticvessel development (Marika Karkkainen, personal communication). VEGFC can regulate physiological and pathological blood-vessel growth in vivo. It is able to stimulate angiogenesis in the mouse cornea and in limb ischaemia29,30. In addition, VEGFC has been shown to regulate the growth of lymphatic vessels in various experimental models. Overexpression of VEGFC in skin keratinocytes leads to dermal lymphatic-vessel hyperplasia16. Signalling by means of VEGFR3 alone was shown to be sufficient for the hyperplasia, as transgenic mice that overexpress a mutant form of VEGFC, which has lost its capacity to bind VEGFR2 and only binds and activates VEGFR3 (VEGFC156S), was able to induce a similar phenotype20. VEGFC was also studied in the mature avian chorioallantoic membrane (CAM), which contains lymphatic vessels that are located mainly around arterioles and veins15. In this assay, VEGFC acts as a highly specific lymphangiogenic factor. However, when VEGFC is applied to the early CAM, in which the lymphatics have not yet developed, it promotes angiogenesis29. The angiogenic versus lymphangiogenic responses to VEGFC might depend on the degree of proteolytic processing of its precursor, and on the expression of its receptors in the blood versus lymphatic endothelial cells of the target tissue. VEGFC also has synergistic effects

LYMPH NODE

A collection of lymphoid cells which occurs along the course of the lymphatics and serves to filter lymph (the fluid of the lymphatics) for antigen presentation and immune-cell proliferation. The lymph node is frequently the initial site to which tumours spread and is therefore an important diagnostic indicator. ANGIOGENESIS

The proliferation of new blood vessels from pre-existing ones. ENDOTHELIUM

The group of cells that line the inner surface of both the blood and lymphatic vessels. Endothelial cells can express specialized receptors that allow interaction with cells of the immune system that are present in the blood and lymph.

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Summary • Tumour metastasis to regional lymph nodes is a crucial step in the progression of cancer. Detection of tumour cells in the lymph nodes is an indication of the spread of the tumour, and is used clinically as a prognostic tool and a guide to therapy. However, the molecular mechanisms that control the spread of cancer to the lymph nodes were unknown until recently. • The proliferation of new lymphatic vessels (lymphangiogenesis) is controlled, in part, by members of the vascular endothelial growth factor (VEGF) family — namely, VEGFC and VEGFD — and their cognate receptor on lymphatic endothelium, VEGFR3. These secreted growth factors are synthesized as propeptides that are activated by proteolysis to form high-affinity ligands that activate VEGFR3 and stimulate lymphangiogenesis. • The recent identification of molecular markers to discriminate between lymphatic endothelium and blood-vessel endothelium has enabled the study of lymphatic vessel formation in experimental models and in human tumours. • Experimental studies with VEGFC and VEGFD have shown that they can induce tumour lymphangiogenesis and direct metastasis to the lymphatic vessels and lymph nodes. By contrast, angiogenic factors such as VEGF act to enhance the growth of tumours by promoting a more extensive blood-vessel supply. • The published patterns of expression of lymphangiogenic factors in human tumours, in general, support the hypothesis that these factors promote the lymphatic spread of human tumours. • The inhibition of tumour lymphangiogenesis, using inhibitory agents that are directed to VEGFC, VEGFD or its receptor VEGFR3 (for example, monoclonal antibodies, receptor bodies or tyrosine kinase inhibitors), could be useful for anti-metastatic approaches to the treatment of human cancer.

| AUGUST 2002 | VOLUME 2

www.nature.com/reviews/cancer


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Embryonic

Pathological

Adult

Stromal cell

Macrophages

Stromal cell

Heart

Lung

Skin

Tumour cell

Supporting cells surrounding vessels

Macrophages

Unprocessed VEGFC/D Proteolytic processing

Angiogenesis

VEGFR2

Lymphangiogenesis

VEGFR2

VEGFR3

Figure 2 | Mode of action of lymphangiogenic growth factors. The lymphangiogenic growth factors vascular endothelial growth factor-C (VEGFC) and VEGFD are produced by a range of cells and tissues during embryogenesis, adult life and certain pathologies, including cancer. The full-length growth factors are processed by as yet uncharacterized proteases that cleave the amino- and carboxy-terminal propeptides (light blue and red L-shapes, respectively) from the VEGF homology domain (VHD) to generate a mature form that consists of dimers of the VHD (purple oval) that bind the receptors VEGFR2 and VEGFR3 with high affinity. In the case of VEGFD, the mature form has a 290-fold and 40-fold greater affinity for VEGFR2 and VEGFR3, respectively, than the unprocessed form33. Activation of VEGFR3 induces lymphangiogenesis, whereas activation of VEGFR2 is thought to drive angiogenesis .

with VEGF during the induction of angiogenesis, and this effect is more prominent in cells that express both of its receptors31. The second lymphangiogenic growth factor to be reported — VEGFD (also known as c-FOS-induced growth factor, or FIGF) — is the most recently discovered member of the mammalian VEGF family17,32. The mature form of VEGFD shares 61% amino-acid sequence identity with VEGFC, and these two growth factors bind to the same receptors on human endothelial cells — namely, VEGFR2 and VEGFR3 (REF. 17; FIG. 1). VEGFD is proteolytically processed in a similar way to VEGFC, and the proteolytic processing seems to regulate VEGFD biological activity and receptor specificity33. Interestingly, in mice, Vegfd binds only to Vegfr3, indicating that Vegfd might have a somewhat different function in mice and humans34. This is uncommon in the VEGF family as these homologous and evolutionarily conserved growth factors are assumed to have similar receptor-binding characteristics in different species.

Mouse Vegfd is able to stimulate the proliferation of vascular endothelial cells and is angiogenic in vitro and in vivo 35. Like VEGFC, human VEGFD is lymphangiogenic when overexpressed in skin keratinocytes20. Little is known about the expression of VEGFD in physiological conditions, but its mRNA has been observed in melanoma cells and fibroblasts, lung mesenchyme and in the adult vascular wall36. The exact roles of VEGFC and VEGFD during embryonic vascular development are likely to be different. In adults, VEGFC and VEGFD might regulate the response of lymphatic vessels in inflammatory processes and in the regeneration of tissues after trauma, but they might also have important roles in physiological and pathological angiogenesis in various conditions. The lymphangiogenic receptor VEGFR3 contains an extracellular domain that consists of seven immunoglobulin homology domains37,38, the fifth of which is proteolytically cleaved, and the resulting polypeptides remain linked by two disulphide bonds39 (FIG. 1). The cytoplasmic domain contains tyrosine kinase domains that are interrupted by a kinase insert sequence. In a recent study, lymphatic endothelial cells were isolated using monoclonal antibodies to VEGFR3 and grown in culture40. Stimulation of VEGFR3 alone protected these cells from serum-deprivation-induced apoptosis and induced cell growth and migration. At least some of these signals were transduced via a protein-kinase-C-dependent activation of the p42/p44 mitogen-activated protein kinase (MAPK) signalling cascade and via AKT phosphorylation40. The culture of lymphatic endothelial cells will allow further analysis of events that are associated with activation of the VEGFR3 lymphangiogenic signalling pathway. Markers of the lymphatic endothelium

Although the lymphatic vessels were discovered approximately 300 years ago41 — at the same time as the blood circulation was described42 — the study of the lymphatic system has remained relatively neglected until recently. This has been, in part, due to the difficulties in recognizing lymphatics in tissues because of morphological criteria, such as their thin walls, compressed lumens (although lymphatics can be distinguished from blood vessels by a lack of pericytes) and a need for specific markers. Therefore, the recent discovery of a range of markers that show specificity for lymphatic endothelium has been a significant advance in the field of lymphangiogenesis (TABLE 1). VEGFR3 was originally thought to be expressed specifically in the lymphatic endothelium, but further studies revealed that it is also expressed in a small subset of blood vessels in normal tissues and, in addition, can be re-expressed in angiogenic blood vessels in certain pathological conditions43,44. Podoplanin is a glomerular podocyte membrane mucoprotein, which occurs together with VEGFR3 in the lymphatic endothelium and in benign vascular tumours and angiosarcomas, but it is also expressed in certain nonendothelial cells, such as osteoblastic cells, kidney podocytes and lung alveolar type I cells45,46. Podoplanin




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Table 1 | Markers exhibiting specificity for lymphatic endothelium within the vasculature Marker

Function

Desmoplakin

Associates with desmosomal Epithelial cells and cardiac muscle cadherins to form a cell-adhesion complex

D6

β-Chemokine receptor

Expressed in a subset of lymphatics only

References 53,95

50

Secondary lymphoid Mediates lymphocyte chemokine adhesion and migration through tissues by acting as a chemoattractant

Expressed in T-cell zones within lymph nodes and lymphoid tissues, and constitutively expressed on the lymphatic endothelium of many tissues

5′-Nucleotidase

Enzyme involved in nucleotide metabolism

Weakly expressed on some blood vessels

LYVE1

Hyaluronan receptor on lymphatic endothelium

Blood sinusoidal endothelial cells in both liver and spleen, and placental syncytiotrophoblasts

48,49, 97,98

Podoplanin

38-kDa membrane glycoprotein of podocytes

Osteoblastic cells, kidney podocytes and lung alveolar type 1 cells

45,46, 99,100

PROX1

Homeobox protein involved in the formation of the lymphatics

Non-endothelial cells in lens, heart, liver, pancreas and nervous system

47,101

VEGFR3

Growth-factor receptor, ligands are VEGFC and VEGFD; signals for lymphangiogenesis

Tumour blood vessels, highly fenestrated blood vessels and macrophages

is present in small lymphatics but not in larger ones that have smooth-muscle cells, whereas all blood vessels and high endothelial venules in the lymph nodes are negative for this protein. PROX1 is a homeobox transcription factor that is involved in the growth and elongation of lymphatic vessel sprouts during development47, and its expression persists in adult lymphatic endothelium. It is also expressed in non-endothelial cells in the lens, heart, liver, pancreas and nervous system. Prox1 +/– mouse embryos develop chylous ascites and die within 2–3 days after birth, whereas Prox1 –/– mice have a completely absent lymphatic system47. Another marker that is located on lymphatic endothelial cells, LYVE1 (lymphatic vessel endothelial hyaluronan receptor-1), is related to the CD44 receptor for hyaluronan. LYVE1 is distributed equally among the luminal and abluminal surfaces of lymphatic vessels and is involved in the uptake of hyaluronan by lymphatic endothelial cells and its transport from the tissues to the lymph48. However, LYVE1 is not completely specific for lymphatic endothelial cells, as it is also present, for example, in normal hepatic blood sinusoidal endothelial cells49. Recently, a β-chemokine receptor D6 was shown to be present in a subset of lymphatic vessels in the skin, intestine and lymphoid tissues50. Interestingly, lymphatic vessels in most other organs remained negative for D6 immunoreactivity. The existence of this receptor on only a subset of lymphatics indicates a functional heterogeneity within the lymphatic vasculature. In addition, the secondary lymphoid chemokine SLC/6Ckine/exodus-2/CCL21 is a useful marker for lymphatic endothelium51. Recent findings revealed coexpression of neuropilin-2 (an isoform-specific receptor for VEGF, VEGFB and placenta growth factor) and VEGFR3 in the lymphatic vessels52. Desmoplakin and 5′-nucleotidase have also been used to distinguish the

576

Sites of expression other than lymphatics

51,96

54

25,43,44, 72,102,103

lymphatic from the blood-vascular endothelium53,54, and as lymphatic capillaries lack a continuous basement membrane, immunohistochemistry for extracellular-matrix components such as type IV collagen and laminin have also been used to distinguish them from blood capillaries55. VEGFR2 is occasionally expressed in lymphatic endothelium56, and the cell-surface-receptor tyrosine kinases TIE1 and TIE2 might also have a role in lymphatic-vessel regulation, as they also appear in lymphatic endothelium56,57 and in mice that are deficient for angiopoietin-2 (Ang2), a Tie2 ligand, that produces a lymphatic phenotype120. In cultured lymphatic endothelial cells and in normal tissues, VEGFR3, LYVE1 and podoplanin were essentially located in the same cells43,49,58. More information needs to be obtained to clarify the specificity of these markers in lymphatic vessels in disease processes. Until then, several markers should be used to confirm the staining of lymphatic vessels in pathological conditions. Lymphatic vessels in cancer

A common clinical finding for many solid tumours, including carcinomas, is the spread of tumour cells through the lymphatics to regional lymph nodes59,60. The detection of this event in the metastatic process is significant for both tumour staging and for designing treatment protocols1,59–61. In particular, the analysis of sentinel lymph nodes has been developed as a method for the diagnosis and staging of a range of cancers, including melanoma and breast cancer 62–64. The question of whether lymphatic vessels actually exist in solid tumours is still debated in the scientific and medical literature12,65–67. The lack of markers to distinguish blood vessels from lymphatic vessels, and the difficulty in identifying these vessels by injection techniques, has meant that, in recent history, the evidence that linked lymphatics in solid tumours with the spread of cancer was not compelling 12.




REVIEWS a Tumour angiogenesis – increased tumour growth Vascular endothelial cells Macrophage

Tumour-cell proliferation

Chemotaxis

VEGFR2

Stromal cell

Tumour cell

Unprocessed VEGF-C/D

b Tumour lymphangiogenesis – increased lymphatic metastasis Lymphatic endothelial cells Tumour-cell invasion

Macrophage Chemotaxis VEGFR2

VEGFR3

Stromal cell

Tumour cell

Figure 3 | Function of lymphangiogenic factors in tumour biology. The lymphangiogenic factors vascular endothelial growth factor-C (VEGFC) and VEGFD are produced by both tumour and stromal cells. a | Processed VEGFC and VEGFD signal through the receptor VEGFR2, stimulating vascular endothelial cells and promoting angiogenesis. This supports the growth and proliferation of tumour cells. b | Processed VEGFC and VEGFD can induce lymphangiogenesis by activating VEGFR3, which stimulates lymphatic endothelial cells. This promotes lymphatic metastasis in experimental tumour models18,19,21 and increases the ability of human cancer cells to metastasize to the lymphatics and local lymph nodes (see TABLE 2). VEGFC and VEGFD also have a role in immune modulation. VEGFC has been shown to be chemotactic for macrophages, which express VEGFR3. Macrophages also express and secrete lymphangiogenic factors, such as VEGFC and VEGFD72,121.

Nevertheless, it has become clear that peritumoral lymphatics, which are often enlarged, are associated with human tumours68, whereas the existence of intratumoral lymphatics has been in doubt 69. It was long thought that lymphatics might be collapsed or could not penetrate into expanding primary tumours because of the high physical pressure inside tumours70. However, recently, intratumoral lymphatics and lymphangiogenesis have been observed19,21,68, and an interesting question is whether or not lymphatic-vessel density, in comparison to blood-vascular density, is related to prognosis and metastatic spread. Role of lymphangiogenesis in cancer spread

Recent work using experimental animal models has highlighted the role of VEGFC and VEGFD in tumour biology (FIG. 3). Transgenic mice that overexpress VEGFC in β-cells of the pancreas (Rip-VEGFC, rat insulin promoter) developed extensive lymphangiogenesis around the endocrine islets of Langerhans18. Furthermore, when tumours were induced in these VEGFC-overexpressing islets — by mating the mice with transgenic mice that express the SV40 T-antigen oncogene in the β-cells (Rip1-Tag2) — metastatic tumour-cell aggregates of β-cell origin were observed in the surrounding lymphatic vessels. These mice also frequently developed metastases in the lymph nodes

that drain the pancreas, whereas tumours in mice lacking the VEGFC transgene did not metastasize, nor were tumour cells observed inside the lymphatic vessels18. VEGFC overexpression did not significantly alter tumour volume, transition from adenoma to carcinoma or tumour angiogenesis — but, interestingly, the tumour incidence was increased, the reasons for which are unknown. Similarly, human breast cancer cells expressing ectopic VEGFC were shown to induce lymphangiogenesis in and around the orthotopically implanted tumours19,71. Nevertheless, VEGFC did not have a significant effect on angiogenesis in these models, although it did promote tumour growth. VEGFC did, however, stimulate angiogenesis in another tumour model72. This apparent discrepancy might be related to the degree of proteolytic processing of VEGFC in each individual tumour model. In a breast cancer model, VEGFC promoted enhanced spreading of tumour cells to the regional lymph nodes, and the degree of tumour lymphangiogenesis correlated with lymph-node metastases73. VEGFC-induced tumour lymphangiogenesis, intralymphatic tumour growth and lymphatic metastasis could be inhibited by adenoviral expression of the soluble extracellular domain of VEGFR371,74. VEGFC expression by a tumour, however, is not always sufficient to induce the formation of functional




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Table 2 | Expression of VEGFC/VEGFD genes in the primary tumours Tumour

Findings in relation to metastasis

References

VEGFC Breast cancer

VEGFC protein expression correlated with lymphatic vessel invasion; diseasefree survival (5 years) of the VEGFC-positive group was significantly poorer*‡

104

Cervical cancer

Multivariate analysis revealed VEGFC mRNA expression to be the sole independent factor influencing pelvic lymph-node metastasis; patients with VEGFC mRNA expression had significantly poorer prognosis*

105

Colorectal cancer

VEGFC mRNA correlated with lymph-node metastasis, lymphatic involvement and invasion depth*‡ Tumours with lymph-node metastasis had higher levels of VEGF expression, but there was no association between VEGFC expression and lymphatic spread*‡

106

Endometrial carcinoma

VEGFC protein expression correlated with vascular invasion, depth of invasion, lymphatic-vessel invasion and lymph-node metastasis‡

108

Oesophageal cancer

VEGFC expression correlated with depth of invasion, tumour stage, venous invasion, lymphatic invasion and lymph-node metastasis‡

109

Gastric cancer

Correlation between grade of VEGFC expression pattern and lymph-node status* VEGFC expression correlated with lymphatic and venous invasion; VEGFC expression had a significant negative impact on the prognosis of patients who did not express VEGF‡ VEGFC protein expression was significantly higher in lymphatic-invasion-positive early gastric cancer (EGC) than that in lymphatic-invasion-negative EGC; also, there was a tendancy for VEGFC to be expressed in node-positive/venous-invasion-positive EGC, compared with node-negative/venous-invasion-negative EGC‡

110 111

107

112

Head and neck squamous-cell carcinoma

Increased expression of VEGF and VEGFC in tumours*; VEGFC expression had predictive value for the presence of cervical nodal metastases

86

Lung adenocarcinoma

Lymph-node metastasis associated with high levels of VEGFC expression; a low VEGFD:VEGFC ratio correlated with lymphatic invasion*

85

Neuroblastoma

No correlation between VEGFC expression and lymph-node metastasis*

113

Non-small-cell lung cancer VEGF and VEGFC expression was associated with nodal microdissemination* VEGFC expression was significantly associated with lymph-node metastasis, lymphatic-vessel invasion and poor outcome‡

114 115

Pancreatic cancer

VEGFC expression was associated with increased lymphatic vessel invasion and lymph-node metastasis, but not with decreased patient survival

116

Prostate cancer

VEGFC mRNA was more abundant in lymph-node-positive patients§

117

Thyroid tumours

VEGFC expression correlates with lymph-node-invasive tumours*

118

Tumours with lymph-node metastasis expressed higher levels of VEGF, but there was no association of VEGFD expression with lymphatic spread*‡ The expression of VEGFD correlated with lymphatic involvement and was found to be an independent prognostic marker for disease-free and overall survival in colorectal carcinoma

107

VEGFD Colorectal cancer

87

Lung adenocarcinoma

Lymph-node metastasis associated with low VEGFD expression*

85

Head and neck squamous-cell carcinoma

Decreased expression of VEGFD associated with cervical-node metastases*

86

*mRNA expression detected by reverse transcription polymerase chain reaction; ‡protein detected by immunohistochemistry; §mRNA expression detected by in situ hybridization; VEGF, vascular endothelial growth factor.

lymphatic vessels66. There might be variation between different tumour types, and the physical pressure exerted by the growing tumour might prevent the growth of lymphatic sprouts into the tumour — an issue that has been debated extensively 69. VEGFD was also shown to promote the metastatic spread of tumour cells via the lymphatics in a mouse tumour model21 (FIG. 3). In addition to lymphangiogenesis and increased metastasis, the tumours that secrete VEGFD also showed an increase of both growth rate and angiogenesis. The growth of the tumour, angiogenesis and formation of metastases were all inhibited by a neutralizing antibody to the bioactive region of VEGFD. The differences between

578

the tumour-angiogenic properties of VEGFC and VEGFD might have been due to differences in their proteolytic processing in the different models. Some of the heterogeneity in the effects of these growth factors could also result from variable expression of their receptors — Vegfr2 and Vegfr3 — on the blood-vascular and lymphatic endothelia. In the above case, in particular, enhanced tumour angiogenesis was probably observed for VEGFD because of its increased proteolytic processing, which resulted in an increased affinity to Vegfr2 (REF. 21), when compared with the VEGFC models. Consistent with this theory, the vascular effects of VEGFC in experimental tumours were reduced when the




REVIEWS mice were treated with blocking antibodies against Vegfr2 (REF. 75). Intratumoral lymphatic vessels were observed in xenograft models19,21,71, but not in a transgenic mouse tumour model18. This could be partially explained by the trapping of vessels in between the rapidly growing tumour foci in the xenografts. On the basis of these observations, tumour-vessel formation can be dissected into pathways that preferentially activate angiogenesis (driven by VEGFR2) and pathways that preferentially activate lymphangiogenesis (driven by VEGFR3), although there is evidence that the receptors share overlapping expression patterns56. Activation of lymphatic endothelial cells by tumour-cell-secreted factors might promote the interaction of tumour cells with lymphatic endothelial cells, and thereby facilitate tumour-cell entry into the lymphatics. In spite of the increased metastatic tendency of VEGFC-overexpressing tumour cells, metastases were seen only in a subset of the tumourbearing mice. Overall, on the basis of studies in which VEGFC or VEGFD have been overexpressed in tumours, it could be that there are additional, rate-limiting steps in the metastatic process 74. The simplest explanation for the metastasis-enhancing effects of VEGFC and VEGFD is that they eliminate one rate-limiting step by increasing the contact surface area between invading tumour cells and hyperplastic lymphatic endothelium. They could also, however, facilitate metastasis by increasing vascular permeability or by changing the adhesive properties or cytokine or chemokine expression patterns of the lymphatic endothelium. VEGFC and VEGFD that are secreted by tumour cells could also have important effects on the tumour interstitial-fluid pressure. VEGFC can increase vascular leakage — but not as efficiently as VEGF — although a parallel increase in lymphangiogenesis could alleviate this effect. The increased interstitial fluid pressure could be a significant determinant of tumour-cell seeding into the blood vascular and lymphatic circulation: recent studies have shown that a proportion of the lumens of tumour blood vessels contain tumour cells76,77. It is not known to what extent tumour-cell-secreted factors are directly responsible for the large lymphatic vessels that are occasionally detected around human tumours. Inflammatory cells, for example, could contribute to the lymphangiogenesis, as VEGFC is chemotactic for macrophages72 and readily induced by proinflammatory cytokines26,27. It is not clear whether the newly formed lymphatic vessels mature in a similar way to the blood vessels, or whether they are more prone to tumour-cell invasion, for example, because of differences in the expression of adhesion receptors. VEGF is known to be able to upregulate the expression of adhesion molecules in the vasculature78, but such a role for VEGFC and VEGFD is not known. Although it seems evident that both VEGFC and VEGFD can induce the growth of new lymphatic vessels, several questions remain unanswered regarding tumour lymphangiogenesis and metastasis. For example, it is not known whether it is sufficient for pre-existing lymphatic

vessels to expand by circumferential growth, or whether new vessels are required for the enhancement of the metastatic process. The intratumoral lymphatic vessels observed are usually collapsed, presumably due to the high physical pressure in solid tumours, which might impair their transport capacity. Also, as in angiogenesis, lymphangiogenesis might occur by several mechanisms, and different regulatory factors might be involved. One example of such a factor is fibroblast growth factor-2 (FGF2), which can promote both angiogenesis and lymphangiogenesis79. VEGFR3 might also be involved in tumour angiogenesis, in addition to its function in tumour lymphangiogenesis. It has been reported to be expressed in blood-vessel capillaries during tumour angiogenesis44,80. Inactivation of Vegfr3 by neutralizing antibodies suppressed tumour growth by destabilizing large blood vessels in xenograft mouse models. Microhaemorrhages were seen in these vessels, indicating that Vegfr3 could be involved in maintaining the integrity of the endothelial-cell lining in the neovasculature81. Frequent Vegfr3 antibody administration was required for the suppression of tumour growth, but the architecture of the non-angiogenic blood and lymphatic vessels remained unaffected. It has also been shown that even a prolonged suppression of Vegf activity in adult mice has no effect on the maintenance of the vascular system, although it suppressed angiogenesis severely in embryos82. The fully established blood and lymphatic vessels seem to be resistant to treatment with these kinds of anti-angiogenic agents. It is still unknown whether VEGFC or VEGFD promotes lymphangiogenesis in human tumours, and, if so, whether or not this increases the rate of metastasis to the lymph nodes. VEGFC expression has been detected in about half of the human cancers analysed83. Several reports have described a correlation between VEGFC expression in human tumours and the formation of metastases in regional lymph nodes. So far, VEGFC levels in primary tumours have been shown to correlate significantly with lymph-node metastases in thyroid, prostate, gastric, colorectal, lung and oesophageal carcinomas (see TABLE 2). Less is known about the presence of VEGFD in human tumours, but this growth factor was shown to be upregulated in human melanomas when compared with melanocytes36. VEGFD was detected in melanoma cells, as well as in vessels that are adjacent to VEGFD-expressing tumour cells, but not in vessels that are distant from the tumours. This indicates that VEGFD binds to the endothelial cells of nearby vessels and contributes in a paracrine manner to the regulation of tumour angiogenesis and lymphangiogenesis — a model that is supported by recent analysis of VEGFD protein and mRNA in non-small-cell lung cancer 84. VEGFD expression, by contrast, was inversely correlated with lymph-node metastasis in lung adenocarcinoma85 and head and neck squamous-cell carcinoma86. Interestingly, expression of VEGFD was recently identified as an independent prognostic marker for both disease-free and overall survival in colorectal carcinoma and correlated with lymphatic involvement for this




REVIEWS

Table 3 | Potential inhibitors of the lymphangiogenic signalling pathway Potential inhibitor

Mode of action

Site of action References

Soluble extracellular domain of VEGFR3 Sequester VEGFC and VEGFD

Extracellular

71,89

Soluble extracellular domain of VEGFR2 Sequester VEGFC and VEGFD; also sequester VEGFA

Extracellular

119

Neutralizing monoclonal antibodies to VEGFC and VEGFD

Block interactions with VEGFR3 and VEGFR2 Extracellular

21,88

Neutralizing monoclonal antibodies to VEGFR3

Block interactions with VEGFC and VEGFD

Cell surface

Peptidomimetics based on VEGFC and/or VEGFD

Block binding of VEGFC/D with VEGFR3

Cell surface

Inhibitors of tyrosine kinase activity of VEGFR3

Block signalling by VEGFR3

Cytoplasm

91,92

Inhibitors of molecules that signal downstream of activated VEGFR3

Block signalling downstream of VEGFR3

Cytoplasm

40

81

VEGF, vascular endothelial growth factor; VEGFR, vascular endothelial growth-factor receptor.

tumour type87. The reason that VEGFD expression correlates with metastasis in some tumours, but has an inverse correlation with metastasis in others, could be due, in part, to different degrees of proteolytic processing of this growth factor in different tumours, or expression of other lymphangiogenic factors. In a recent study, proliferating intra-tumoral lymphatics were observed in discrete hotspots in a subset of human head and neck squamous-cell carcinomas and correlated with lymphnode metastasis — at least in the case of oropharyngeal carcinoma68. These intratumoral lymphatics were immature in morphology with small lumens and comprise only a small number of endothelial cells. Although, in this study, the presence of these proliferating intratumoral lymphatics did not seem to correlate with VEGFC expression, further analysis will be required to thoroughly assess the role of VEGFC and VEGFD in the regulation of these vessels. Inhibition of lymphangiogenesis

LYMPHOEDEMA

A condition in which fluid drainage from tissue is insufficient due to a dysfunctional or overloaded lymphatic system.

580

Despite advances in surgery, radiotherapy and chemotherapy, the prognosis of many cancers remains poor, so new approaches for anti-metastatic therapy would be most welcome. Inhibitors that block the VEGFC/VEGFD/VEGFR3 signalling pathway that controls tumour lymphangiogenesis might have the potential to block lymphogenous metastatic spread. Monoclonal antibodies (mAbs) that antagonize the interaction of VEGFC and VEGFD with the ligand-binding domain of VEGFR3 would be useful inhibitors of this pathway (TABLE 3). It was recently reported that a neutralizing VEGFD mAb (VD1) that blocks interactions with Vegfr2 and Vegfr3 (REF. 88) inhibited angiogenesis, lymphangiogenesis and metastatic spread via the lymphatics in a mouse tumour model21. A mAb against Vegfr3 that blocked the binding of Vegfc induced micro-haemorrhage from tumour blood vessels in a mouse cancer model — although the effects on tumour lymphatics were not analysed81. One alternative to antibodies would be to use a soluble version of the extracellular domain of VEGFR3 to sequester VEGFC and VEGFD. A study in which a transgenic mouse expressed the extracellular domain of VEGFR3 in skin keratinocytes under the

control of the keratin 14 gene promoter showed that depletion of the ligands for VEGFR3 with this domain inhibited the formation of lymphatic vessels in the embryo89. Additionally, adenoviral-mediated delivery of the soluble receptor domain blocked the growth of peritumoral lymphatic vessels in a mouse model of breast cancer71 and inhibited lymph-node metastasis, but not lung metastasis, in a lung cancer model74. Another approach for inhibiting the lymphangiogenic signalling pathway would be to use orally active small molecules that interfere with the binding of VEGFC/VEGFD to VEGFR3, or that inhibit the tyrosine kinase catalytic domain of this receptor. The potential of such approaches has been illustrated by recent studies showing that inhibitors of the tyrosine kinase catalytic domain of the closely related VEGFR2 block tumour angiogenesis — at least in animal models90,91. Interestingly, a set of indolinones have been described that inhibit the catalytic activity of VEGFR3, but not VEGFR2 (REF. 92). The effects of these compounds in vivo, however, have not yet been reported. One potential complication of targeting the VEGFR3 signalling pathway is that lymphatic-vessel function in normal tissues could be effected. Little is known about the role of VEGFR3 in the maintenance and function of mature lymphatics. It will therefore be crucial to carefully monitor the effects of anti-lymphangiogenic approaches — especially those that target the VEGFR3 signalling pathway — on the integrity and function of mature lymphatics in normal adult tissues. Clinical use of pro-lymphangiogenesis

An important function of the lymphatics is to regulate the pressure of interstitial fluid in tissues by transporting excess fluid back into the circulation. Oedema represents an imbalance between lymph formation and its absorption into the lymphatic vessels. Clinical situations in which the lymphatic system is involved include LYMPHOEDEMA due to impaired lymphatic drainage. Lymphoedema can be hereditary (primary lymphoedema; Milroy’s disease) or caused by inflammatory or neoplastic obstruction of the lymphatic vessels, including accumulation of ascites




REVIEWS fluid due to peritoneal carcinomatosis or oedema of the arm following surgery or radiotherapy for breast cancer (secondary lymphoedema). Lymphoedema is treated, at present, by manual lymphatic drainage and by compressive garments. The discovery of specific genes that are involved in the regulation of lymphatic vessels, and in the pathology of lymphoedema, should make the design of targeted treatments for this condition possible. Several mouse models have recently shown that VEGFC and VEGFD can be used to stimulate lymphangiogenesis and are, therefore, potential treatments for lymphoedema16,20,52,93. Such therapy could be used in non-hereditary, regional forms of lymphoedema that result from surgery or lymphaticvessel destruction after cancer therapy. As VEGFR3 signalling has a role in lymphatic endothelial-cell survival58,94, long-term growth-factor expression might be needed to induce lymphangiogenesis and maintain these vessels in patients with chronic lymphoedema. The functional characteristics of the newly formed lymphatic vessels — such as their connections to draining lymphatic vessels — still require additional studies. Future directions

Blood and lymphatic vessels are formed in an interactive

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manner during embryonic development, but both vessel types are maintained in a rather quiescent state in adults and are active only in sites of new tissue growth. Angiogenesis has been extensively studied for over a decade, but lymphangiogenesis is a relatively new topic for vascular biology. As lymphatic vessels are important for the spread of cancer, lymph-node metastasis is an effective prognostic indicator, and partly determines the therapeutic approaches for cancer treatment. The discovery of molecules that are involved in the biology of lymphatic vessels, lymphatic-specific markers and techniques for culturing lymphatic endothelial cells has enabled a more extensive study of the many roles of the lymphatics in developing tissues and cancer. VEGFC and VEGFD both stimulate the growth of lymphatic vessels, being the first growth factors found for lymphatic endothelial cells. Encouraging results have recently been observed from animal models in which blocking the VEGFC/VEGFD/VEGFR3 signalling pathways led to inhibition of metastatic spread of tumour cells via the lymphatic vasculature. Conversely, stimulation of this signalling pathway shows promise for the treatment of lymphoedema. Clinical trials using these strategies will reveal the potential of anti-lymphangiogenics in treating human cancer.

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Acknowledgements M.G.A. and S.A.S. are supported by the National Health and Medical Research Council of Australia and the Anti-Cancer Council of Victoria (ACCV). M.E.B. is supported by an ACCV postdoctoral fellowship. We thank J. Stickland for assistance with production of the figures.

Online links DATABASES The following terms in this article are linked online to: Cancer.gov: http://www.cancer.gov/cancer_information/ breast cancer | colorectal cancer | gastric cancer | head and neck squamous-cell carcinoma | lung cancer | melanoma | non-small-cell lung cancer | oesophageal carcinoma | oropharyngeal carcinoma | prostate cancer | thyroid cancer GenBank: http://www.ncbi.nih.gov/Genbank/ SV40 T antigen LocusLink: http://www.ncbi.nlm.nih.gov/LocusLink/ AKT | Ang2 | CCL21 | CD44 | β-chemokine receptor D6 | type IV collagen | desmoplakin | FOS | keratin 14 | laminin | LYVE1 | MAPK | neuropilin-2 | placenta growth factor | Prox1 | PROX1 | TIE1 | TIE2 | VEGF | VEGFB | Vegfc | VEGFC | Vegfd | VEGFD | Vegfr2 | VEGFR2 | Vegfr3 | VEGFR3 Access to this interactive links box is free online.

