Carcinogenesis vol.27 no.9 pp.1729–1738, 2006
doi:10.1093/carcin/bgl031
Advance Access publication April 5, 2006
REVIEW
Lymphatic vessels in cancer metastasis: bridging the gaps
Ramin Shayan, Marc G.Achen and Steven A.Stacker
Brief history of the lymphatic system in cancer
Angiogenesis Laboratory, Ludwig Institute for Cancer Research,
PO Box 2008, Royal Melbourne Hospital, Victoria 3050, Australia
To whom correspondence should be addressed
Email: [email protected]
The French surgeon Le Dran (1) first noted in the sixteenth
century that cancers of the breast which spread to axillary
lymph nodes had significantly worse survival outcomes than
those that were localized only to the primary tumour. Some
200 years later, Halsted (2) set about performing sometimes
disfiguring radical excisions of both the primary breast cancer
and the metastatic lesions in the axillary lymph nodes (3). The
next major clinical development regarding the lymphatics in
cancer occurred in the 1950s when clinicians began to use
radioisotope injections to better understand which regional
lymph node groups drain different parts of the body (4),
essential information for identifying potential routes of cancer
metastasis via the lymphatic vasculature.
The 1990s saw the adaptation of lymphatic mapping for the
prediction of each patient’s lymphatic drainage from a specific
tumour, and for identification of the ‘sentinel lymph node(s)’
(i.e. the lymph node(s) most likely to contain spreading cancer
cells) within the lymphatic drainage basin (5). The same decade also yielded the discovery of lymphatic-specific molecular
markers to identify lymphatic vessels, which were hitherto
histologically indistinguishable from blood vessels (6). The
advent of these techniques led to the realization that lymphatics
are fundamental to cancer metastasis and many other pathological processes, and has generated intense clinical and
scientific interest in the lymphatic vasculature as a potentially
valuable therapeutic target (7).
Distant organ metastasis is the most important factor in
determining patient survival in cancer. This is thought to
occur via the body’s own systems for transporting fluid and
cells, the blood vascular and lymphatic systems. Cancer
cells may exploit these vascular systems by expressing
growth factors, which alter the normal pattern of angiogenesis and lymphatic vessel growth (lymphangiogenesis), thus
creating conduits for tumour metastasis. With respect to
lymphatic metastasis, techniques which allow the mapping
of a tumour’s lymphatic drainage and sampling of the
‘sentinel node’ from the regional lymph node group provide
crucial prognostic information, determine further treatment and offer a window into tumour–host immune interactions. Aberrant drainage patterns so identified are both
clinically significant, and highlight important anatomical
and molecular complexities not explained by existing
models of lymphatic development or anatomy. The molecular controls of tumour lymphangiogenesis and factors
determining which lymphatic vessel subtypes are induced
may be targets for novel therapeutics designed to restrict
cancer metastasis. Furthermore, analyses of these control
mechanisms will enhance our understanding of the interactions between the tumour cells and the lymphatic vasculature. For many years, disparate groups of clinical
researchers and basic scientists have been working to
unravel the mysteries of the lymphatic system. This review
aims to summarize these contributions, in terms of the
history, identification, structure and function of lymphatic
vessels in cancer and the role they play in tumour metastasis. Current ideas about the roles of lymphangiogenic
growth factors, their signalling pathways in lymphatic
metastasis and therapeutic opportunities to restrict this
spread will also be explored.
Abbreviations: E, embryonic day; ECM, extracellular matrix; LEC,
lymphatic endothelial cell; MAPK, mitogen activated protein kinase; PC,
proprotein convertase; PTK, protein tyrosine kinase; RTK, receptor tyrosine
kinase; SMC, smooth muscle cell; VEGF vascular endothelial growth factor;
VEGFR vascular endothelial cell growth factor receptor.
Structure, function and development of the lymphatic system
The lymphatic vessels are integral for interstitial fluid volume
regulation and absorption of dietary fat. In addition, the lymphatics are important for immune function and in the metastatic
spread of cancer (8). The intricate network of thin-walled vessels which constitutes the lymphatic vasculature commences
within the superficial dermis of the skin as highly permeable
blind-ending sacs, and are referred to as ‘lymphatic capillaries’
or ‘initial lymphatics’ (Figure 1) (8,9). Fibrillin filament
anchors between lymphatic endothelial cells (LECs) and the
extracellular matrix (ECM) translate interstitial fluid volume
expansion into lateral displacement, to create temporary intercellular fenestrations (10). The lack of a continuous basement
membrane surrounding the initial lymphatics facilitates entry
of fluid into these vessels and restoration of normal interstitial
volume, resulting in a slackening of the fibrillin filaments, and
eventual return of the LECs to their overlapping resting position. Studies by Weber et al. (11) suggest that ECM signalling
stimulates additional cytoskeletal structural alterations, which
further aid fluid influx.
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R.Shayan, M.G.Achen and S.A.Stacker
Fig. 1. Schematic representation and molecular characteristics of lymphatic vessel subtypes found in the dermal and subcutaneous layers of normal
mammalian skin. The initial lymphatic capillaries are blind-ending vessels within the superficial layers of the dermis, which collect fluid and cells for
transportation to the pre-collecting vessels. The pre-collecting vessels are located in the deeper layers of the dermis and are characterized by the appearance
of valves, a basement membrane and SMCs within their structure. Once vessels enter the subcutaneous fat, they exhibit more uniform valves, circumferential
SMC coverage, a thicker more complex wall and a continuous basement membrane. These so-called ‘collecting ducts’ constitute the major route for return of
fluid to the cardiovascular system. Molecular markers for LECs are useful to detect and differentiate these vessel subtypes, and to characterize the functional
role they play in lymphangiogenesis and cancer metastasis. Note that not all molecular markers are shown on the LECs.
Pre-collecting lymphatic vessels, located in the deep dermis,
drain fluid from the initial lymphatics (Figure 1). They have
segments which contain valves and are surrounded by a basement membrane and smooth muscle cells (SMCs) (12). These
segments alternate with other regions that are morphologically
akin to the initial lymphatic capillaries. The pre-collecting
lymphatics, in turn, drain into the ‘collecting lymphatics’,
which are located in the subcutaneous tissue. These vessels
have circumferential SMCs and regular intraluminal valves
(13). Those collecting lymphatics which are >200 mm in
diameter also have arterial-type mural intima, media and
adventitia (13); however, do not normally possess pericytes
(14). They propel lymph at an average of 10 mm/s by a combination of intrinsic wall motion (generated by specialized
pacemaker-SMCs), and compression by adjacent arterial
pulsation and skeletal muscle contraction (15–17), eventually
returning lymph to the central veins. The collecting lymphatics
coalesce into lymphatic trunks, then intrathoracic ducts.
Unaided by ‘lymphatic hearts’, found in animals such as
toads (18), the larger mammalian lymphatic vessels generate
surges of up to 100 mm/s several times each minute to drain
fluid from most tissues to a lymph node within 20 min (19–21).
1730
Alterations to flow may be mediated by nitric oxide synthase in
LECs, acting via nitric oxide to produce SMC dilatation
(22,23) and may be under autonomic control, as illustrated
by splanchnic stimulation influencing thoracic duct flow
(24). Recent work by He et al. (25) indicates that these lymphatic vessels may also dilate in response to tumour-derived
growth factors, hence increasing the lymph flow and capacity
to transport tumour cells to lymph nodes.
Lymph fluid passes through a series of lymph nodes or other
secondary lymphoid organs (gastrorespiratory submucosal
lymphoid aggregations such as Peyer’s patches and tonsils)
(26). Afferent lymph node ducts divide before passing beneath
the capsule of the node into cortical sinuses then pass through a
reticuloendothelial cell filter (26). Antigen-presenting cells display antigen epitopes to lymphocytes within these organs, triggering them to clonally expand and tailor an individualized
‘antigen-specific’ immune response (27). Whilst lymph continues through the medullary sinus to the hilar region of the lymph
node and into efferent ducts, tumour cells may become trapped
and proliferate here or spread further to distal organs (21).
The first lymphatic vessels emerge in the mouse at about
embryonic day (E) 10.5, when endothelial cells sprout from the
Lymphatic vessels in cancer metastasis
anterior cardinal vein to form the primary jugular lymph sacs
(28). These endothelial cells express the lymphatic transcription factor Prox-1, which is essential for lymphatic development and is thought to be a major determinant of LEC fate (28).
These cells also express the receptor tyrosine kinase (RTK)
vascular endothelial growth factor receptor-3 (VEGFR-3), the
cell surface receptor that binds the lymphangiogenic growth
factors VEGF-C and VEGF-D. It is the nearby expression of
VEGF-C which initiates the first sprouting of lymphatics from
the anterior cardinal vein, via VEGFR-3 stimulation (29). The
primary jugular lymph sacs subsequently also sprout to form a
primitive lymphatic plexus, which spreads throughout the head
and neck, thorax and forelimbs (8). The lymphatics continue to
evolve from E12.5 onwards, via a series of posterior lymphatic
sacs which are derived from local veins, and progress to penetrate interstitial tissues nearby (28). They form a primitive
dermal plexus by E15.5 (28), and continue to develop after
birth into a deep dermal pre-collecting layer, from which
sprout the beginnings of a subepidermal lymphatic capillary
plexus (30). The LECs of the latter lymphatic vessels differ
from those of the deep dermal lymphatics both morphologically and in terms of the cell surface molecular markers
expressed (Figure 1) (30). The timing of the formation of
the initial lymphatic capillary layer also varies depending
on the location of the skin, but occurs after the second postnatal day in the mouse (30). The endothelium of mature lymphatic vessels expresses the cell surface markers podoplanin and
LYVE-1, although LYVE-1 appears to be more abundant in
lymphatic capillaries than in larger vessels (Figure 1) (30). In
contrast, EphrinB2, a cell surface tyrosine kinase, is expressed
on LECs in pre-collecting lymphatic vessels, where it is essential for the maturation of these vessels after birth, and on
collecting lymphatic vessels, but is not expressed on LECs
in lymphatic capillaries (30) .
Relatively little is known about development of lymph
nodes or other lymphoid tissues. These structures arise from
connective tissue protruding into embryonic lymph sacs, which
later forms the first lymph node anlagen tissue at the predetermined site of future nodes (26,27). This involves differentiation of mesenchymal cells into stromal organizer cells and
aggregates of CD45+CD4+CD3+ lymphoid ‘tissue inducer
cells’ (27). The two cell types interact within the anlagen to
induce expression of adhesion molecules on stromal organizer
cells and release homeostatic chemokines such as CCL21,
CCL19 and CXCL13. These in turn attract further lymphoid
‘tissue inducer cells’ and other hemopoietic cells (27,31).
Interestingly, studies in mutant mice have demonstrated that
lymph node development is genetically distinct from that of the
lymphatic vasculature (32).
The involvement of the lymphatics in cancer
The initial spread of cancer cells is seen histologically as
tumour escape beyond a defined boundary and may include
the invasion of local lymphatic vessels (33). Micrometastasis
to lymph nodes, thought to occur before most primary tumours
are clinically detectable, often heralds distant organ metastasis
at the time of diagnosis or after some period of delay, and is
therefore the most significant prognostic indicator in many
human cancers (34). Hence, lymph node staging may alter
the type and timing of treatment offered to the patient, and
it is critical that all nodes potentially containing metastatic
tumour cells are sampled surgically for histological analysis
(34). Failure to detect occult metastasis may be responsible for
around one-third of early stage colorectal cancer patients who
were originally classed as lymph node-negative on histopathology, later developing systemic or recurrent disease (35,36).
‘Sentinel lymph node biopsy’ is one method of reliably
sampling lymph nodes to which tumour cells have metastasized. Originally developed for melanoma (37), it has since
been adapted for application to several other malignancies
(38,39), in particular, breast carcinoma (40). It is a procedure
in which a pre-operative map of lymph node(s) draining a
primary cancer is obtained by injection of radiolabelled colloid
(41). The sentinel node(s) is then located intraoperatively with
a hand held gamma probe (42), in conjunction with visualization of blue dye injected at the commencement of the procedure (43). This enables accurate identification and surgical
excision of the sentinel lymph node(s), deemed to be those
node(s) most likely to harbour metastatic cancer cells (5,37). If
histological analysis of the sentinel lymph node(s) demonstrates the presence of metastatic cells, surgical clearance of
the remaining lymph nodes within the same anatomical area is
required for further histological staging and prognostication.
Furthermore, histologically positive sentinel lymph nodes usually pre-date distant metastasis and are, therefore, an indicator
of poor prognosis (5,37), even if they occur outside the
regional lymph node group (44,45). Detection of positive sentinel lymph node(s) results in reclassification of a patient to a
group with higher risk of recurrence, which may make them
eligible for systemic adjuvant anti-metastasis treatment and
thus potentially result in a survival benefit (36,46–48). In contrast, the absence of metastatic cancer cells within the sentinel
node is taken to indicate disease-free status in the remaining
lymph nodes in the nodal group (49), and the patient is consequently spared the morbidity of further lymphablation surgery, which could lead to undesirable side effects such as
lymphoedema (50). Ablation of metastatic lymph nodes and
focussed external beam radiation therapy also aid local disease
control, as misdiagnosis or inadequate clearance of metastatic
nodes may result in bulky, unresectable disease (51).
The precise mechanisms by which tumour cells move preferentially towards particular lymph node(s) remain poorly
defined. It was originally envisaged that lymphatic invasion
took place passively as an advancing tumour front eroded the
walls of any vessels in its path and that metastasis occurred by
passive drainage (52); however, recent evidence indicates that
a more complex interaction between tumour cells and the
lymphatic endothelium may take place (53). The availability
of markers with specificity for LECs has enabled the identification and quantification of previously undiscernible lymphatic vessels in human cancer (54), and other pathological states
(55,56).
The main ‘workhorse’ reagent for identification of lymphatic vessels, since the late 1990s, has been anti-LYVE-1 antibody (6,57,58). LYVE-1 is a CD44 homologue protein which
is involved in hyaluronan and immune cell transport (6,57),
and may itself be implicated in tumour cell trafficking to lymph
nodes (59). Heterogeneous LYVE-1 expression on cultured
and tumour-induced LECs (59), and differential expression
between normal lymphatic vessel subtypes (Figure 1) (30),
means that a combination of LEC molecular markers are
required to reliably identify different lymphatic vessels (9).
Podoplanin is a mucin-type transmembrane glycoprotein,
which despite being expressed on kidney podocytes,
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R.Shayan, M.G.Achen and S.A.Stacker
osteoblasts and Type I alveolar cells is expressed uniformly
across the lymphatic vessel subtypes and has thus proven to be
another useful LEC marker (60–62). Whilst its specific function remains elusive, mutant mice lacking podoplanin display
lymphatic defects which contribute to respiratory compromise
and death, but do not interfere with normal blood vasculature
(63). Podoplanin expression is regulated by the homeobox
transcription factor Prox-1, which, whilst not widely used to
identify cancer lymphatics, has been important for understanding lymphatic development during embryogenesis (64,65).
VEGFR-3 was amongst the earliest markers used to characterize lymphatic vessels (66); however, the observation of
VEGFR-3 expression on blood vessels in tumours and
wound granulation tissue has meant that it is less appropriate
for specific identification of lymphatics in these conditions
(67,68). Finally, molecules recently identified on lymphatic
vessels, such as EphrinB2 and EphB4, which are responsible
for blood vessel territorial demarcation, are differentially
expressed between lymphatic vessel subtypes (Figure 1)
(30), and may therefore become useful in distinguishing different lymphatic vessels in cancer.
Whilst immunohistological studies using lymphatic-specific
markers have demonstrated the existence of proliferating
intratumoral lymphatic vessels in several types of human
tumour, the functional significance of intratumoral lymphatic
vessels remains controversial (69). It has been proposed that
intratumoral lymphatics are not able to transport tumour cells
because the elevated hydrostatic pressure within a tumour may
compress these vessels (69,70). Certain studies have reported
intratumoral lymphatic density as an accurate independent
predicted of poor disease-free survival, and of increased metastatic propensity in such important human tumours as melanoma, and carcinoma of the breast, endometrium, colon, lung,
prostate, ovary, pancreas and in the head and neck (71–79).
Peritumoral lymphatics are the lymphatic vessels immediately surrounding a tumour. It has been suggested that these
may represent pre-existing vessels compressed into a peritumoral rim by the expanding tumour mass (80). However,
LECs in peritumoral lymphatics have been observed to be proliferating around some human cancers, suggesting that these
vessels can also arise due to lymphangiogenesis (71–74). Additionally, peritumoral lymphangiogenesis was significantly associated with regional metastasis and poor disease-free and overall
survival in malignant melanoma (81), and detection of lymphangiogenic growth factors was also shown to be of prognostic
significance in human carcinoma of the cervix, ovary, breast and
gastrointestinal tract (74,75,77,78,82–85). Although the relative importance of intratumoral versus peritumoral lymphatics
for metastatic spread remains a subject of debate, it is clear that
the lymphatic vessels associated with a tumour can be important
for metastasis and patient outcome (86). Further, experimental
and clinopathological studies indicate that lymphangiogenesis
can contribute to the formation of these lymphatic vessels (86),
therefore, an understanding of the molecular control of this
process may identify novel therapeutic targets for restricting
the spread of cancer (7).
Molecular regulation of lymphangiogenesis in cancer
VEGF-C and VEGF-D constitute the lymphangiogenic ‘subfamily’ of the VEGF growth factors, and have become important predictors of tumour growth, lymphangiogenesis and
1732
metastatic behaviour (85). All of the members of the VEGF
family (the others being VEGF-A, placenta-derived growth
factor and VEGF-B) are related by a common VEGFhomology domain (VHD), which contains a 30% conserved
amino acid sequence and a characteristic cystine-knot motif
(87). Unique to the two-member lymphangiogenic ‘subfamily’, N-terminal (N-pro) and C-terminal (C-pro) propeptides (87,88) are proteolytically cleaved from the VHD by
enzymes including plasmin (89) and ‘proprotein convertases’
(PCs) (88,90). This process generates mature, bioactive forms
of VEGF-C and VEGF-D, which consist of VHD dimers and
display enhanced affinity for VEGFR-3 (88,91), and thus make
this group of enzymes important to the tumorigenicity of
cancers which secrete these growth factors (92,93). Developmentally, VEGF-C is vital for normal lymphatic embryogenesis (87), a finding supported by transgenic and gene
deletion animal models (55,94). Additionally, exogenous
administration of both VEGF-C and VEGF-D were sufficient
to rescue lymphatic vessel sprouting (29), and transgenic
expression of either ligand alone induced lymphangiogenesis
independent of angiogenesis (95,96).
VEGFR-3 is a major transducer of lymphangiogenic signalling, acting predominantly through phosphorylation of Akt,
a PI-3 kinase-dependent serine-threonine kinase, and a PKCdependent p42/p44 mitogen activating protein kinase (MAPK)
(94,96,97). VEGFR-3 activation not only prevents LEC
apoptosis in culture but also stimulates proliferation, migration
and cell survival under serum deprivation conditions via this
pathway (98).
Experimental models of lymphangiogenesis in cancer
The VEGF-C expressing MCF-7 human breast cancer cell line
demonstrates increased rates of lymph node metastasis in the
presence of increased intratumoral, and particularly peritumoral lymphatics (99). Furthermore, soluble antibody to
VEGFR-3 was shown to inhibit lymphangiogenesis and subsequent metastasis (99). A similar phenomenon of increased
intratumoral and peritumoral lymphatic vessels was seen in an
alternative VEGF-C expressing breast cancer line, MBAMD-435 (100). The enhanced access to lymphatics was also
associated with an increase in regional lymph node spread, and
particularly pulmonary visceral metastasis (100). Consistent
with Kukk et al.’s work (101), however, which showed the
unprocessed predominantly VEGFR-3-specific 31 kD form of
VEGF-C (as produced by these breast cancer models) to be
insufficiently processed to stimulate VEGFR-2, significant
angiogenesis did not occur in either setting (99,100). In contrast, the VEGF-C overexpressing MeWo human melanomaderived cell line caused increased proliferation of both blood
and lymphatic vessels, both intratumorally and peritumorally,
due to increased proteolytic processing of VEGF-C (102).
Enlarged neolymphatics in and around the VEGF-C expressing
AZ521 human gastric cancer model resulted in lymph node
metastasis in 95% of AZ521-VEGF-C mice, compared with
29% of controls (103), and similar findings were demonstrated
in the normally benign pancreatic b-cell tumours, when
implanted in VEGF-C overexpressing transgenic mice (104).
In the case of VEGF-D, growth factor expressing, stably
transfected 293 EBNA tumours xenografted into SCID-NOD
mice demonstrated active roles in both tumour growth and
lymphangiogenesis, with a concomitant increase in regional
lymph node metastasis, compared with their VEGF-A expressing counterparts (105). Whilst VEGF-A promoted growth and
Lymphatic vessels in cancer metastasis
angiogenesis, neither lymphangiogenesis nor nodal dissemination were enhanced, and empty vector controls produced well
contained, indolent tumours (105). Lymph node metastasis rates
occurred in a VEGF-D dose-related fashion, and monoclonal
antibodies raised to the bioactive region of VEGF-D competitively antagonised mature VEGF-D binding of VEGFR-2 and
VEGFR-3, and inhibited tumour metastasis (106).
Detmar et al. (107) compared a VEGF-A producing,
GFP-expressing, K14 promoter-driven transgenic mouse
model with a non-VEGF-A expressing control, in which squamous cell carcinoma was generated by multistep, topical chemical
induction. Interestingly, not only did VEGF-A appear to play
a role in tumour growth but also stimulated both angiogenesis
and lymphangiogenesis, apparently signalling via an upregulated VEGFR-2 on LECs (107).
Overall, these animal models correlate with findings in several human tumours. Significant tumour-induced lymphangiogenesis has been noted in human melanoma samples, and
importantly, were directly related to the risk of lymph node
metastasis and patient survival (46,71). Of note, several
authors have found that human breast carcinoma samples
seem to demonstrate metastasis in the absence of intratumoral
lymphangiogenesis (69,86,108). Human breast cancer specimens in which intratumoral vessels were often collapsed and
poorly staining with proliferation markers, but in which peritumoral lymphatics were increased and contained tumour
emboli (86), may indicate particular significance of the peritumoral microenvironment in tumour lymphatic proliferation
and metastatic activity. They may also illustrate the technical
limitations of using single lymphatic markers, such as
LYVE-1, alone (69), as they may fail to stain relevant vessels
if downregulated in the particular lymphatics subtypes formed
(30). Consistent with the regression to the primitive embryological state often seen in pathological contexts, blood vessel
VEGFR-3 expression is observed on immunohistochemical
studies of both benign and malignant vascular tumours (Kaposi
sarcoma, 80% of spindle cell hemangiomas, 80% of angiosarcomas), non-vascular tumours, (including melanomas, carcinomas, other sarcomas) and perivascular tumours (109).
These findings may implicate VEGFR-3 in maintaining the
endothelial integrity during tumour angiogenesis, which generates vessels more akin to primitive, rather than mature blood
vessels (68).
Targeting lymphatics vessels for clinical benefit
Therapeutics
The VEGF-C/VEGF-D–VEGFR-3 pathway. Therapeutic
approaches for the inhibition of RTKs such as VEGFR-3,
include monoclonal antibodies, small molecule inhibitors, peptide drugs and antisense techniques (7,110,111). Folkman’s
vision of anti-angiogenesis as a cancer treatment has been
realized with the release of an anti-VEGF agent for the
treatment of metastatic colorectal carcinoma (112,113). Analogous to VEGF-A blockade to reduce tumour angiogenesis
(114,115), an approach to block VEGFR-3 ligands VEGF-C/
VEGF-D is promising for the inhibition of tumour lymphangiogensis and lymphogenous metastasis (105,106,116). Hence,
Achen et al. (106) raised monoclonal antibodies to the VHD of
human VEGF-D which bind both unprocessed and fully processed VHD with high affinity, and inhibit their binding to both
VEGFR-2 and VEGFR-3.
Overall, experimental models demonstrating suppression of
VEGFR-3 signalling have shown inhibition of both peritumoral
and intratumoral lymphangiogenesis, angiogenesis and metastatic spread (116,117). Administration of soluble VEGFR3-immunoglobulin fusion protein, which binds VEGF-C and
blocks VEGFR-3 signalling, and intravenous recombinant
adenoviruses expressing VEGFR-3-immunoglobulin, both
induce regression of tumour-induced lymphatic vessels (116).
In addition, the interference with ligand–receptor interactions
and the resulting inhibitory effect on lymphangiogenesis and
metastasis produced by adeno-associated virus-delivered soluble VEGFR-3 decoy receptor was dose-related in VEGF-C
secreting PC-3 and A375 tumour models (117). Further evidence of the therapeutic potential of soluble VEGFR-3 was
provided by transgenic expression of soluble VEGFR-3 in
mouse skin, which inhibited fetal lymphangiogenesis, and
induced regression of already formed lymphatic vessels, though
the blood vasculature was unaffected (93). These transgenic
mice develop a lymphoedematous phenotype, characterized
by foot oedema and dermal fibrosis. They survive the neonatal
period despite almost complete absence of lymphatic vessels in
several tissues, which later regenerate (94). Similarly, missense
mutations interfere with VEGFR-3 signal transduction to cause
primary (congenital) lymphoedema (56).
Proteolytic processing of VEGF-C/VEGF-D
VEGF-C expressing tumours which induced angiogenesis and
lymphangiogenesis, but were inhibited by mutating the proVEGF-C cleavage site, demonstrated that processing of proVEGF-C is crucial to receptor activation, and thus generation
of its biological effects (93,118). The enzymes responsible
include the PCs and the serine protease, plasmin, which has
been shown to cleave both C-propeptides and N-propeptides
from the human VEGF-C and VEGF-D VHD, to promote
lymphangiogenesis and cancer metastasis (89). As a potent
proteolytic enzyme which is active at cleaving both propeptides of the lymphangiogenic growth factors, the parallel role
that plasmin plays in fibrinolysis, a process which is crucial in
regulating blood clotting, may help coordinate lymphangiogenesis in the later stages of tissue repair. Promisingly, alterations to specific PC growth factor binding sites have been
shown to inhibit PDGF-A precursor processing by furin, the
phosphorylation of its cognate RTK, and the resulting cellular
proliferation (119). Furthermore, specific PC blockade in
several stably transfected, growth factor secreting tumour
models has been found to reduce tumour growth, malignant
phenotype and migration (93,118). Thus, inhibitors of the PCs
and plasmin, which restrict the cleavage of VEGF-C and/or
VEGF-D, may also constitute novel agents for the treatment of
human tumours and/or in adjuvant therapy to prevent tumour
growth, metastasis or recurrence (93,118,119).
Intracellular signalling cascades
The majority of work in this area has focussed on inhibiting
tumour angiogenesis (110,120,121); however, blockade of
intracellular signalling cascades may also prove to be useful
for suppression of downstream VEGFR-3 signalling, as many
of the VEGF family of RTKs employ common signalling
mechanisms (97,98,120,122,123). In particular, PI-3 kinaseAkt and PKC-p42/p44 MAPK signalling cascades are the pathways activated by growth factor stimulation of VEGFR-3
(94,96–98), and may represent potential therapeutic targets
for inhibiting lymphangiogenesis induced by tumour-secreted
1733
R.Shayan, M.G.Achen and S.A.Stacker
growth factors (110,121). Several small molecular agents targeting cytoplasmic protein tyrosine kinase (PTK) in preclinical
models inhibit angiogenesis and tumour progression, and
have reached clinical trials (124). One example is SU11248
(sunitinib or Sutent), which has reached Phase III clinical
trials, and offers targeted treatment in select tumours such
as carcinoma of the lung, renal cell carcinoma, and
gastrointestinal tumours (125–127). Other PTK inhibitors
which target VEGFR-2 and VEGFR-3 pathways, also currently involved in clinical cancer treatment trials include
CEP-7055 (128), PTK 787/ZK 222584 (129,130) and BAY
43-9006 (131). The latter is a Raf-1 kinase inhibitor, which
demonstrates anti-angiogenesis and anti-tumour activity via
MAPK signalling and VEGFR tyrosine kinase blockade in
preclinical tumour xenograft models, and is being trialed for
use in a range of human tumour settings, including such
common cancers as carcinomas of the breast, prostate, lung,
pancreas, kidney, thyroid, pancreas and ovary (131,132).
Chemokines and adhesion molecules
Certain receptor–ligand relationships between tumour and host
tissues, and soluble factors, known as chemokines, which regulate hemopoietic cell migration, may be ‘hi-jacked’ by cancer
cells to facilitate the location of and entry into lymphatic
vessels, and their movement along them towards lymph
nodes (133). Furthermore, tumour–endothelial interactions
may be responsible for particular cancers displaying metastatic
affinity towards specific tissues, and chemokine–ligand guided
interactions may create a chemical gradient which predisposes
circulating metastatic cells to home toward, and settle within a
certain distant organ tissue (133).
A prime example in lymphatic metastasis is CCL21/SLC, a
ligand expressed on LECs for lymphocyte and dendritic cell
signalling, and guidance towards lymph nodes via the CCR7
receptor (134–136). Human melanoma and breast cancer cell
lines expressing CCR7 have demonstrated increased affinity for
lymphatic endothelium and resulted in increased lymph node
metastasis in animal models, a process which was inhibited by
the administration of neutralizing anti-CCL21 antibodies (137).
The CXCR4–CXCL12 pathway is also a well-characterized
T-lymphocyte signalling mechanism, and CXCL12 is differentially expressed between individual colorectal carcinomas, providing a prognostic factor for local recurrence, liver metastases
and poor overall survival (133). Interference with CXCR4 signalling may present a further target in disease-directed therapy
for colorectal carcinoma (133).
Expression of adhesion molecules, which normally participate in immune cell traffic between interstitial and vascular
compartments, may also be important in tumour entry to, or
exit from the lymphovascular space during metastasis (138).
Bevacqua et al. (138) demonstrated an in vitro correlation
between attachment potential and metastatic propensity in
malignant melanoma and breast carcinoma cell lines, using
adhesion assays with human tumour cells. One particular adhesion molecule, L-selectin, forms a ligand–receptor pair with
mannose receptor, which is expressed on lymphatic endothelium (139). It orchestrates the intravasation of circulating
lymphocytes from interstitial tissues into lymphatic vessels,
and subsequent homing to lymph node high endothelial venule
addressins, a process which may be exploited by L-selectin
expressing tumour cells, in order to promote lymph node metastasis, and which has been inhibited in animal studies by
administration of anti-L-selectin monoclonal antibody (53).
1734
Both mannose receptor and common lymphatic endothelial
and vascular endothelial receptor (CLEVER)-1 have been
implicated in tumour metastasis via adhesion of malignant
cells to lymphatic endothelium (140), in some human cancers,
and represent further potential anti-metastatic therapeutic targets (141). Proinflammatory cytokines and the resulting
immune cell aggregations may also stimulate additional
lymphangiogenic growth factors, and so provide an indirect
method of inhibiting lymphatic vessel proliferation in the
region of a tumour (142,143).
Diagnostics
Imaging localization of lymphatic drainage pathways
The cutaneous lymphatic drainage pathways from the site of a
primary tumour may be highly variable between patients, even
within the same areas of the body (144). Up to 30% of these
tumours therefore defy clinical predictability of which lymph
nodes, in which regional node groups, may contain cancer cells
(145), and contradict long-held anatomical ideas of drainage
patterns (146).
Significant examples detected using lymphatic mapping technology include drainage (and potential tumour spread) from
superficial back skin to deep lymph nodes in paraaortic and
retroperitoneal areas; across the midline; or from periumbilical
skin through the chest wall (147,148). These routes bypass the
classical drainage pathways through regional lymph nodes and
may involve multiple nodal fields (149). Such cancers would be
erroneously classified as lymph node-negative using conventional methods (147,150), in 14–37% of patients with melanomas on their limbs, trunk, or head and neck (147–152).
Future directions for lymphatic imaging will include the
ongoing development of lymphatic mapping for cancers in
deeper viscera such as the gastrointestinal and respiratory
tracts (48). The next challenge is to develop methods of sentinel lymph node assessment that are non-invasive, yet as
accurate as present methods. Adaptations using labelled antibodies or other tracers with specificity for lymphatic vessels or
lymph nodes, could assist in analysing the levels at which
abnormalities of lymphatic drainage occur, whether they
involve unrecognized pattern variations in ‘collecting lymphatics’, or ‘lymphaticovenous’ or ‘interlymphatic subtype’
shunts, and whether aberrant lymphatic channels may be influenced by tumour-derived growth factors.
Conclusion
Understanding lymphogenous tumour metastasis represents a
crucial step in the treatment of human cancer. Since the development of lymphatic markers, several diverse disciplines may
now converge in an attempt to define the respective roles
played by lymphatic vessels and tumour cells and products
in facilitating metastasis to lymph nodes and beyond. Understanding the complexities of lymphatic development, anatomy
and pathophysiology in terms of the influences of lymphangiogenic growth factors, receptor signalling and tumour
immunomodulation may yield an array of new therapeutic
targets for application in the treatment of cancer.
Acknowledgements
We thank Janna Taylor and Diana Keshtiar for their assistance in generating the
figure illustrations, and R.S. would also like to acknowledge the ongoing
Lymphatic vessels in cancer metastasis
supervision and guidance of Prof. G. Ian Taylor, AO. S.A.S. is supported by a
Senior Research Fellowships from Pfizer and M.G.A. by a Senior Research
Fellowship from the National Health and Medical Research Council of
Australia. The Angiogenesis Laboratory is supported by funds from the
NH&MRC. R.S. is a recipient of the Raelene Boyle Sporting Chance Foundation and Royal Australasian College of Surgeons Cancer Research Scholarship.
Conflict of Interest Statement: S.A.S. and M.G.A. are shareholders in
Lymphatix Ltd and have received funding from Imclone Systems Inc.
S.A.S. also holds Pfizer Australia fellowship.
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Received January 30, 2006; accepted March 20, 2006