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Historically, lymphatic vessels were considered passive participants in tumor metastasis by simply providing channels for tumor cells to transit to draining lymph nodes. The discovery of several key lymphatic-specific molecular markers and an increased availability of in vitro and in vivo experimental systems to study lymphatic biology have however highlighted a much more complex, active role for the lymphatic vasculature in metastatic tumor spread. This review will briefly describe the lymphatic system and lymphangiogenesis and then focus on the role of the lymphatic system in cancer metastasis. The progression of our understanding from the lymphatic system as a somewhat passive conduit for metastasis to an active participant in metastatic tumor dissemination, regulated by a complex array of lymphangiogenic factors, chemokines, and immune cell subsets, will be described.
Metastatic spread of tumor cells to distant organs is the leading cause of mortality from cancer.1,2 Although metastatic tumor spread can occur via a variety of mechanisms including direct local invasion of tissue or the seeding of body cavities, most metastases arise following invasion of and dissemination via the circulatory systems. While both the blood and lymphatic vascular systems have been implicated, preclinical experimental systems supported by clinical evidence suggest the most common pathway of initial metastasis is through the lymphatic system.3,4 Indeed, in many human cancers, the detection of tumor metastases in the tumor-draining lymph node (LN) is the first step in tumor dissemination and is one of the most important markers for both patient prognosis and therapeutic strategy decisions.
Historically, lymphatic vessels were considered passive participants in tumor metastasis by simply providing channels for tumor cells to transit through. However, the discovery of several key lymphatic-specific molecular markers and an increased availability of in vitro and in vivo experimental systems to study lymphatic biology have highlighted a much more complex, active role for the lymphatic vasculature in metastatic tumor spread. Here, we review how our understanding of the role of the lymphatic system in cancer metastasis has evolved from a relatively passive model to an active system regulated by a complex array of lymphangiogenic factors, chemokines, and immune cell subsets.
The lymphatic vasculature primarily functions to regulate tissue fluid homeostasis, collect antigens and other macromolecules from peripheral tissues, and traffic immune cells such as antigen-presenting dendritic cells from the periphery to lymph nodes.5-8 The lymphatic vascular network provides a unidirectional transport system that unlike the blood vascular system lacks a central pump, thereby relying on skeletal muscle contraction and respiratory movement for the transport of lymph. Importantly, the lack of a central driving force within lymphatic vessels provides a transport network for cells in which shear stress is minimal and cell survival correspondingly optimal.9 The lymphatic vasculature begins as thin-walled lymphatic capillaries that start blind ended in the peripheral tissues and are structurally optimal for the absorption or uptake of fluids, proteins, and cells. Indeed, lymphatic capillaries are lined with a continuous single-cell layer of endothelial cells with a discontinuous basement membrane and, in contrast to blood vessels, are not encircled by pericytes or smooth muscle cells. Moreover, the presence of tight and adherens junctions between lymphatic endothelial cells is rare, with the majority of interendothelial cell interactions maintained by “button-like” junctions. These overlapping junctions render peripheral lymphatic capillaries highly permeable to interstitial fluids and proteins and also facilitate immune cell transmigration.10
Lymphatic capillaries are connected to surrounding tissue by anchoring filaments that extend deep into the adjoining tissue to firmly attach lymphatic endothelial cells to extracellular matrix fibers. These anchoring fibers act to ensure opening of the usually closed capillaries when tissue pressure increases, enabling protein-rich lymph fluid and immune cells to enter the lymphatic vascular system.11-13 Lymph that enters peripheral lymphatic capillaries initially drains to precollecting lymphatic vessels, which eventually merge into larger lymphatic vessels surrounded by a basement membrane and lymph flow–promoting smooth muscle cells, while one-way valves prevent retrograde lymph flow.12,14 All lymph trafficked through collecting lymphatic vessels passes through several sequential lymph nodes prior to collection in the thoracic duct and subsequently returns to the venous circulation at the junction of the jugular and subclavian veins (Fig. 1).
The unique structural architecture of the lymphatic vasculature therefore facilitates both the absorption of tissue fluids and trafficking of immune cells. Unfortunately, invasive tumor cells can exploit the loose, overlapping endothelial cell junctions and incomplete basement membrane of lymphatic capillaries to take advantage of the transport network that is highly amenable to cell survival. It has become apparent that lymphangiogenesis, the generation of new lymphatic vessels,15-17 actively contributes to tumor metastasis.
Lymphangiogenesis is a dynamic process during embryogenesis but is largely absent under normal physiological postnatal conditions. Indeed, in the adult, lymphangiogenesis only takes place during certain pathological conditions such as inflammation, tissue repair, and tumor growth.6,7,18 Under pathological conditions, a major contribution has been established for the proliferation and sprouting of new vessels from pre-existing lymphatic vessels.15,16 The relative contribution to new vessels from circulating endothelial progenitor cells17 remains unclear.
The relatively recent identification of several key lymphatic-specific molecular markers and factors that promote lymphatic vessel growth has propelled our understanding of the lymphatic vasculature in both physiological and pathological situations. The first, and most comprehensively studied, prolymphangiogenic factors identified were vascular endothelial growth factor (VEGF)–C and –D, which bind to a tyrosine kinase receptor, VEGF receptor (R)–3, expressed on the lymphatic endothelium.19-22 Genetic mouse models have demonstrated that VEGF-C but not VEGF-D23,24 is required for lymphatic development during embryogenesis. Conversely, targeted overexpression of VEGF-C or VEGF-D in the epidermis of transgenic mice resulted in lymphatic hyperplasia in the skin.25,26 Although deletion of VEGFR-3 is embryonic lethal due to cardiovascular failure,27 evidence suggests that inactivation of VEGFR-3 results in lymphedema in both mice and humans.28,29
Both VEGF-C and VEGF-D are initially produced as prepropolypeptides, and upon stepwise proteolytic processing to their fully mature forms, binding affinities for both VEGFR-3 and VEGFR-2 increase.30,31 VEGFR-2 expression on the lymphatic and blood endothelium therefore enables both growth factors to also exert lymphangiogenic and angiogenic effects.32,33 The demonstrated prolymphangiogenic activity of VEGF-A in tumor model systems, which binds to VEGFR-2 and not -3, further supports a role for VEGFR-2 in lymphangiogenesis.34-36 Neuropilin receptor-2, originally identified in nervous system development, also acts as a coreceptor for VEGF-A, -C, and -D. Neuropilin receptors (Nrps) have very short intracellular domains and are not known to activate any signaling pathways or exhibit enzymatic activity. Originally, Nrps were reported to enhance VEGF receptor signaling by enhancing the binding between VEGF ligands and receptors.37,38 However, more recent studies have suggested Nrps may function independently of VEGF receptors, acting as modulators of endothelial cell migration.39,40 Importantly, neuropilin-2–deficient mice also show impaired lymphatic development characterized by abnormal patterning and a marked reduction in small lymphatic vessels and capillaries.41
More recently, several other factors with prolymphangiogenic activity have been identified. These include hepatocyte growth factor, which binds to the c-met receptor,42 angiopoietin-1 together with its endothelial cell–specific receptor Tie-2,43,44 fibroblast growth factor-1 and -2,45-47 platelet-derived growth factors,48 insulin-like growth factor-1 and -2,49 adrenomedullin,50 and endothelin-1.51 The recent identification of transforming growth factor-β1 as a negative regulator of lymphangiogenesis52 further demonstrates the complexity that underpins the molecular regulation of lymphangiogenesis.
Tumor-induced lymphangiogenesis is mediated by lymphangiogenic growth factors that are produced and secreted by the tumors themselves, stromal cells, tumor-infiltrating macrophages, or activated platelets.53-56 In recent years, experimentation has focused on the role of VEGF-C and VEGF-D in cancer progression. The overexpression of either VEGF-C or VEGF-D in tumors significantly increased tumor-associated lymphatic vessel growth (primarily at the tumor margin) and increased incidence of lymph node metastasis (Fig. 1).15,57-59 Conversely, blockade of signaling via VEGFR-3 using either soluble VEGFR-3 fusion proteins that act as a trap for VEGF-C and -D,59-61 or neutralizing antibodies targeting VEGFR-3,62,63 reduced tumor lymphangiogenesis and lymphatic metastasis. Additionally, a role for VEGF-A in tumor-mediated lymphangiogenesis and metastasis has also been described.64 Transgenic mice overexpressing VEGF-A in the skin exhibited enhanced tumor lymphangiogenesis and metastasis to lymph nodes and beyond when subjected to a multistep chemical skin carcinogenesis cancer model.64 Taken together, these murine tumor experimental systems suggest that tumors can actively induce the growth of lymphatic vessels and that this growth promotes metastasis to lymph nodes.
In addition to increasing lymphatic vessel density, lymphangiogenic growth factors also act to enlarge and dilate lymphatic vessel size.65-67 Interestingly, VEGFR-2 activation is the key mediator of vessel enlargement, contrasting activation via VEGFR-3 that appears more critical for lymphangiogenic sprouting.68 Enlarged lymphatic vessels that drain the tumor enhance the delivery of tumor cells to the lymph node most probably via enhanced lymphatic flow rates69 and facilitation of metastatic tumor cell cluster dissemination.65,67,70
A role for neuropilin-2 in tumoral lymphangiogenesis and metastasis has also recently been described. Neuropilin-2, normally expressed in lymphatic vessels during embryogenesis,41 has recently been found expressed in tumor-associated lymphatic vessels in both experimental mouse tumor models71 and human skin cancers.72 Importantly, functional blockade of neuropilin-2 using a neutralizing antibody blocked lymphatic endothelial cell migration but not proliferation, reduced tumoral lymphangiogenesis, and delayed metastasis to sentinel lymph nodes.71 The localized expression of neuropilin-2 on tumor-associated but not on normal preformed lymphatic vessels suggests that this receptor may be an alternative target to prevent lymph node metastasis via the lymphatic vessels.71 Experimental tumor model systems have therefore established that tumor-induced increases in the lymphatic vasculature, mediated by prolymphangiogenic factors, correspond with increased metastasis to lymph nodes and possibly beyond to distal organs.
A great number of studies have investigated the role of lymphangiogenic factors in human cancer metastasis primarily via immunohistochemical analysis of tumor tissue samples and retrospective comparison to patient outcomes.9,73,74 A number of studies have indicated that increased lymphatic vessel invasion significantly increases the risk of lymph node metastasis, distant metastasis, and death. With regards to breast cancer, lymphatic vessel invasion (LVI) positively correlated with metastasis to sentinel nodes.75-77 Specifically, the presence of peritumoral LVI significantly correlated with lymph node metastasis, being 3-fold more prevalent in node-positive versus node-negative patients.78 Peritumoral LVI was also identified as a key prognostic indicator for the survival outcomes of patients with breast cancer.79
Many human tumors of diverse tissue origins show elevated expression of VEGF-C and/or VEGF-D.80-82 Moreover, the expression of these lymphangiogenic growth factors has been correlated with lymphatic vessel density (LVD), lymphatic metastasis, and disease outcome.54,73,83-85 Furthermore, increased lymphatic expression of VEGFR-3 in patients with breast cancer correlated with increased metastasis-containing lymph nodes and reduced patient disease-free and overall survival.86 It is important to note that not all studies have described a correlation between elevated VEGF-C or VEGF-D and either increased LVD, lymph node metastasis, or survival.85,87 These differences highlight the complexity that underpins the interplay between the lymphatic vasculature and malignant tissues, and imply multiple factors and mechanisms regulate lymphatic metastasis. The majority of clinical studies do however demonstrate that a correlative relationship exists between the expression of lymphangiogenic factors and overall poor patient prognosis.
Tumor-associated lymphatic vessels, although sharing some biological markers and regulatory mechanisms similar to those involved in physiological lymphangiogenesis, likely display differing molecular profiles to normal lymphatic vessels.71,88 Comparison of the RNA profiles of cultured lymphatic endothelial cells isolated from the vasculature of normal tissue, compared to those isolated from highly malignant VEGF-C–expressing fibrosarcoma, revealed significant differences in the expression of more than 790 genes.88 Several genes that encode components of endothelial junctions, subendothelial matrix, and vessel growth/patterning were upregulated (e.g., endothelial-specific adhesion molecule [ESAM], CD105, and the immunoinhibitory receptor CD200), while reduced expression of subendothelial matrix proteins including collagens, fibrillin, and biglycan was also described. Importantly, ESAM-positive tumor-associated lymphatics were correlated with lymph node metastasis in carcinoma patients. These studies suggest that in addition to providing additional vasculature for tumor cells to disseminate through, tumor-associated lymphatics may exhibit distinct molecular profiles that potentially enhance tumor progression and metastasis. Moreover, the finding that many other genes involved in biological processes such as inflammation and chemotactic migration were also differentially regulated suggests that an even greater number of processes may be differentially regulated in tumor-associated versus normal lymphatic function. The impact of these differentially expressed genes upon metastatic dissemination and tumor progression remains to be determined.
Lymphangiogenesis can occur both at the tumor periphery and within the malignant cell mass. Although a role for peritumoral lymphangiogenic vessels has been described in tumor metastasis, it is not clear if the same is true for intratumoral vessels. Intratumoral vessels are often collapsed or occluded by infiltrating tumor cells and thereby possibly nonfunctional.89 Indeed, using transplanted VEGF-C–overexpressing B16-F10 melanomas, 40% of peritumoral but not intratumoral vessels were shown to be functional.90 Although this study failed to demonstrate lymphatic flow from the peritumoral vessels, subsequent studies using B16-F10 melanoma have demonstrated increased lymph flow to draining lymph nodes when compared to non–tumor-bearing controls.91,92 Importantly, VEGF-C–over expressing fibrosarcomas exhibited a further increase in volumetric lymphatic flow (when compared to control tumors) that correlated with increased tumor cell dissemination and increased lymph node metastasis.93 Conversely, blockade of VEGF-C reduced lymphatic hyperplasia, lymph flow, and lymph node metastasis.94
Recent experimental studies have identified a novel mechanism of lymphatic metastasis whereby tumors exert lymphangiogenic effects in sentinel lymph nodes prior to metastatic tumor dissemination.64,95 Employing a multistep chemical carcinogenesis regimen in transgenic mice with epidermal overexpression of either VEGF-A or VEGF-C resulted in significantly increased primary tumor-associated lymphangiogenesis. Most surprising, however, was the observation that lymphangiogenesis within the lymph node preceded the arrival of metastatic cells within the node. VEGF-C– and VEGF-A–mediated lymphangiogenesis is further enhanced within tumor-bearing lymph nodes, resulting in increased lymphatic flow and further induction of lymphangiogenesis in distant nodes (Fig. 1). Importantly, evidence from this model suggests that VEGF-C promotes metastasis not only to draining lymph nodes but also beyond to distant tissues.95 Remarkably, an absence of lymph node metastases resulted in no detection of distant organ metastases, further highlighting the critical role for lymphatic metastasis in tumor dissemination. These findings, supported by the detection of similar changes in the axillary nodes of breast cancer patients prior to evidence of metastasis,96 suggest that lymphangiogenic factors may act to prepare a premetastatic niche conducive to the establishment of secondary tumors. The distal lymphangiogenic effects mediated by VEGF-C prior to metastasis imply that lymphangiogenic growth factors may systemically induce alterations in tissue stroma not only within the primary tumor but also in the draining lymph nodes, secondary lymphoid organs, and other distant metastatic sites. These findings therefore have important implications for the identification of a potentially broader, more diverse role for lymphangiogenic factors in metastatic tumor dissemination than initially anticipated.
One of the key physiological roles of the lymphatic vasculature is the trafficking of immune cells from peripheral tissues to lymph nodes, where adaptive immune responses are instigated. This process is actively regulated and controlled by a family of secreted proteins called chemokines. Under normal physiological conditions, chemokines are critical to the coordinated homing of hematopoietic cells to specific sites and tissues.97,98 Tumor cells, through the expression of chemokine receptors, exploit this established lymphatic trafficking system to mediate both invasion into the lymphatic vasculature and beyond to form metastases in lymph nodes and distal tissues. The initial identification of the chemokine receptors CXCR4 and CCR7 on human breast cancer cells and the expression of their respective ligands (CXCL12 [SDF-1] and CCL21) in organs that tumors normally metastasize to99 have led to a rapid expansion in the field of chemotactic tumor metastasis.
CXCR4 and its ligand CXCL12 (also referred to as stromal cell–derived factor -1 [SDF-1]) are widely expressed in normal tissues and play fundamental roles in fetal development, mobilization of hematopoietic stem cells, and trafficking of lymphocytes.100 In inflamed mouse skin, the CXCR4/CXCL12 chemokine axis promotes lymphatic trafficking of dendritic cells, indicating that lymphatic vessels likely engage dendritic cells via the production of CXCL12.101 A great number of tumor cells of diverse tissue origins have also been found to express CXCR4.102 Importantly, CXCR4 is largely absent from normal breast, prostate, or ovarian epithelia; however, it is characteristically expressed on malignant epithelial cells isolated from these tissues. The mechanisms by which malignant cells upregulate expression of functional CXCR4 are not clearly understood; however, studies have implicated hypoxia-induced upregulation of CXCR4.103,104 Under normoxic conditions, the tumor suppressor von Hippel-Lindau (VHL) negatively regulates CXCR4 via its capacity to target hypoxia-inducible factor (HIF) for degradation. However, under hypoxic conditions, a characteristic feature of many solid tumors, this process is suppressed, resulting in HIF-mediated upregulation of CXCR4.103 Malignant cells that express functional CXCR4 exhibit directed metastasis into tissues that express CXCL12.105 A high concentration of CXCL12 in the lymph nodes and the ensuing gradient this creates within lymphatic vessels strongly attract tumor cells, resulting in increased metastases to the node (Fig. 2).106-108 Tumor-associated lymphatic vessels, but not normal lymphatic vessels, highly express CXCL12, highlighting an active role for the tumor-associated lymphatic endothelium in metastatic tumor spread.72
Evidence suggests that dissemination of metastases to distal organs beyond the draining lymph node is also governed by a CXCL12 gradient. Indeed, tissues that highly express CXCL12, such as the lung, liver, and bone, are preferential sites for metastasis.99,109,110 The CXCR4/CXCL12 axis has also been found to play a role in metastatic dissemination of chemoresistant melanoma.111 CXCL12 secreted by lymphatic endothelial cells within metastatic sites was found to mediate the targeted recruitment of this chemoresistant, CXCR4-CD133–positive population of malignant cells. Importantly, blockade of CXCR4 signaling, when combined with chemotherapy, impaired lymph node and pulmonary metastases and impaired tumor growth. This study indicates that an activated lymphatic endothelium might provide a niche that promotes dissemination of chemoresistant melanoma.111 Activation of the CXCR4/CXCL12 axis has also recently been identified as a key player in the lymphatic invasion and metastasis of malignant cells in patients with extramammary Paget disease.72 Paget cells undergoing epithelial-mesenchymal–like transitions were closely associated with lymphatic invasion. Invasive cells demonstrated elevated CXCR4 expression, while subcapsular sinusoidal lymphatic endothelial cells and macrophages expressed CXCL12 prior to the arrival of metastatic cells within the tumor-draining lymph node.72
CXCR4/CXCL12 axis antagonists have been investigated as potential therapeutic reagents in preclinical mouse models. Anti-CXCR4 treatment inhibited the spread of breast cancer xenografts to sentinel lymph nodes,99 while organ metastases were inhibited when CXCL12-blocking antibodies were employed in a model of non–small cell lung cancer.110 In addition to the demonstration that such an approach may prove therapeutically beneficial for patients, these studies also highlight a crucial role for the functional activation of both tumor cells and the lymphatic endothelium for effective metastatic tumor spread (Fig. 2).
The chemokines CCL19 and CCL21 are also key players in the active metastatic dissemination of malignant cells via the lymphatic system. These chemokines are the only known ligands for the chemokine receptor CCR7 and under physiological conditions regulate the trafficking of CCR7-positive T and B lymphocytes and antigen-presenting dendritic cells to lymph nodes. CCL21 is produced and secreted by fibroblast reticular cells and high endothelial venules of secondary lymph nodes,112 while fibroblast reticular cells may be the only source of CCL19 within the lymph node.113 Both CCL19 and CCL21 are secreted by lymphatic endothelial cells114,115 and are also found expressed in nonlymphoid organs including the lung, colon, stomach, and heart. The role of the CCR7/CCL21/CCL19 axis in normal physiology and immunity has been comprehensively reviewed.116 This chemokine network plays key roles not just in tissue-specific homing of lymphocytes but also in the organization of thymic architecture, lymphoid neogenesis, and as a key regulator in the balance between immunity and peripheral tolerance. These roles and how they relate to lymphatic-mediated metastasis will be discussed in greater details in a following section.
CCR7 expression on tissue sections of cervical, colorectal, gastric, mammary, esophageal, lung, and prostate carcinomas has been correlated with metastasis to lymph nodes.99,117-120 Using experimental models, CCR7-positive tumor cells have demonstrated chemotactic migration towards CCL21-producing lymphatic endothelial cells121 and metastasis to tumor-draining lymph nodes (Fig. 2).122 Moreover, blockade of CCL21 using a soluble inhibitor was also shown to inhibit metastatic migration in vivo.123 In addition to sensing chemotactic gradients established by lymphatic endothelial cells, tumor cells may also set up autologous gradients via secretion of CCL19 and CCL21 into the extracellular matrix.124 This secretion in the presence of slow interstitial flow may preferentially guide tumor cells towards more, rather than less, functional lymphatic vessels.124 The biophysical properties of the lymphatic microenvironment are therefore linked with the active chemotactic migration of tumor cells into the lymphatic vasculature, highlighting the dynamic processes that regulate lymphatic metastasis.
Increased lymphatic flow was reported to upregulate CCL21 expression by both the lymphatic endothelium125 and also the lymph node paracortex, primarily by T cell zone fibroblastic reticular cells,126 and thereby may further enhance the migration of metastatic cells into the lymphatics. Flow-mediated upregulation of CCL21 increased transmigration of dendritic cells, suggesting flow-regulated chemotactic enhancement may also be important for the active migration of CCR7-positive metastatic cells into the lymphatics (Fig. 2). This is particularly important when considering the previously discussed increases in lymphatic flow associated with tumor-draining lymph nodes compared to more distal nondraining nodes.91,92 Active metastatic spread to the lymphatic vasculature may be further enhanced by a recently identified cross-talk mechanism that links VEGF-C and CCL21.127 Tumor cell expression of both VEGF-C and CCR7 was found to synergistically promote tumor invasion into the lymphatics, primarily mediated by VEGF-C–induced increases in lymphatic endothelial cell expression of CCL21.127 These studies describe interrelated mechanisms that exist between lymphangiogenic growth factors and metastatic chemokines. Moreover, they suggest that VEGF-C may play a more diverse role within the tumor microenvironment and at distal sites such as the tumor-draining lymph node than previously anticipated. In addition to mediating active tumor invasion of lymphatics, VEGF-C may also affect CCR7-expressing immune cells and thereby potentially regulate tumor immunity. Indeed, emerging evidence suggests that lymphangiogenesis, an activated lymphatic endothelium, and the key growth factors and chemokines involved may play a critical role in the tumor microenvironment, the draining lymph nodes, and the resulting tumor-immune responses generated.
The host immune system recognizes and interacts with tumors and can be dramatically affected by molecules that originate from within the tumor microenvironment. The studies from our group that describe the regional effects of both VEGF-A and VEGF-C on lymphangiogenesis in sentinel lymph nodes prior to lymph node metastasis64,95 are one such example of tumor-derived factors influencing draining lymph nodes. The expansion of the lymphatic network within the node, primarily the sinusoids, provides a premetastatic niche for tumor cells. Emerging evidence suggests that this niche may not only be structurally more amenable to the arrival of tumor cells but that these changes may also influence the immunological phenotype both within the node and in the peritumoral region where new lymphatic structures are formed. Indeed, tumor-derived factors can influence both innate and antigen-specific immune functions. Multiple mechanisms may act to suppress tumor-specific immune responses in patients, including increased intratumoral and circulating suppressor cell populations such as myeloid-derived suppressor cells and T regulatory lymphocytes, polarization of the cytokine milieu from a cytotoxic Th1 to an immunosuppressed Th2 phenotype, diminished presentation of tumor-derived antigens, and the outgrowth of poorly immunogenic tumor cell populations.
The sentinel lymph node, being the direct drainage site for the tumor, is strongly influenced by tumor-derived factors. Sentinel lymph nodes are also the point of contact for tumor-derived antigens and immune cells and are therefore extensively studied for detection of early immunological changes that may facilitate tumor progression. Many studies have identified alterations in both local and regional immune functions that may contribute to the expansion and metastasis of tumors and importantly have highlighted that these changes can in fact occur prior to metastasis.128 This immune modulation is driven, at least in part, by factors derived from the tumors. Among these potentially immunomodulatory factors are the lymphangiogenic growth factors VEGF-A and VEGF-C and the chemotactic ligand CCL21. Although many of the published studies are largely correlative and do not directly link lymphangiogenesis with immune modulation, these preliminary findings provide new insight into potential novel roles for tumoral and lymph node lymphangiogenesis in metastasis.
VEGFs may impact tumor-immune surveillance via modulation of dendritic cell maturation and recruitment. Host professional antigen-presenting cells such as dendritic cells are key to tumor-specific immune responses. Dendritic cells sample and present tumor-derived antigens to naive T cells via major histocompatibility complex (MHC) class I or class II molecules and thereby initiate antigen-specific antitumor-immune responses. Inadequate presentation of tumor antigens by dendritic cells is therefore one potential mechanism by which tumors escape from the host immune system. Tumor-produced VEGF-A can directly modulate dendritic cell differentiation. VEGF-specific neutralizing antibodies abrogated the negative effects of tumor cell–conditioned media on the differentiation of dendritic cells from hematopoietic progenitor cells.129 Moreover, recombinant VEGF-A administration to naive mice inhibited dendritic cell development and was associated with an increase in the proportion of myeloid-derived suppressor cells,130 well-documented suppressors of antitumor immunity.131 Immature dendritic cell populations that arose in the presence of high levels of VEGF-A have been shown in coculture experiments to stimulate T cells to exhibit an immunosuppressive phenotype characterized by expression of CD25 and CTLA-4 and the secretion of TGF-β, VEGF-A, and IL-10.132 Importantly, however, blockade of VEGF-A using a neutralizing antibody recovered the function of dendritic cells in tumor-bearing mice.133 Moreover, in gastric cancer patients, immunohistochemical staining of carcinoma tissues revealed an inverse correlation between tumor-infiltrating dendritic cells and the expression of VEGF-A.134 The observed decrease in tumor-infiltrating dendritic cells was correlated with an overall poor patient prognosis.134 Tumor-expressed VEGF-C has also been suggested to impact dendritic cell infiltration into gastric carcinoma.135 Increased VEGF-C expression negatively correlated with the degree of dendritic cell infiltrate into the tumor, suggesting that VEGF-C may negatively impact the recruitment of dendritic cells.
VEGFs have been shown to modulate T cell proliferation, phenotype, and activity. Indeed, small interfering RNA-mediated downregulation of VEGF-C in a mouse mammary tumor model has been shown to not only decrease tumoral lymphangiogenesis but also to impact tumor-infiltrating lymphocytes.136 Specifically, downregulation of VEGF-C significantly decreased the number of CD4+ cells infiltrating the tumor, while both CD8+ cell and CD11b-CD11c+ dendritic cell numbers increased. Moreover, tumor-associated leukocytes isolated from tumors with reduced levels of VEGF-C exhibited higher levels of concanavalin A–induced proliferation.136
These findings are supported by recent studies that demonstrate VEGF-A can also modulate T cell activity, specifically via direct inhibition of proliferation. In vitro proliferation assays using circulating T cells from both ovarian cancer patients and healthy donors demonstrated that VEGF-A directly inhibited T lymphocyte proliferation. VEGFR-2 was upregulated upon T cell activation, and further experiments utilizing anti–VEGFR-2–blocking antibodies showed that VEGF-A–mediated proliferation suppression was mediated directly through VEGFR-2 activation. VEGF-A also significantly reduced the cytotoxic activity of T cells.137 These data are supported by findings that VEGF-A can polarize peripheral blood lymphocytes from a Th1 to a Th2 phenotype.138 Th1 cells are characterized by their ability to secrete the proinflammatory cytokine IFN-γ that is key to the survival, proliferation, and effector function of cytotoxic T cells. Polarization to the potentially immunosuppressive IL-4–secreting Th2 phenotype can have a significant impact upon the generation of effective cytotoxic T cell antitumor immunity and thereby provide a permissive environment for tumor progression and metastasis. Importantly, a shift from Th1 to Th2 immune polarization phenotypes has been correlated with poor prognosis in patients with adenocarcinoma.139
VEGF-A has also been implicated in the induction and maintenance of T regulatory cells.140,141 Increased tumor-derived VEGF-A expression resulted in elevated numbers of tumor-infiltrating T regulatory cells in murine melanoma and colon carcinoma models,140 while coculture of VEGF-A–overexpressing colon carcinoma with peripheral blood mononuclear cells increased the proportion of T regulatory cells.141 T regulatory cell populations from cancer patients were found to express VEGFR-2,142 specifically on a subset characterized by high foxp3 expression that also exhibited a strong immunosuppressive phenotype.142 This study implicates VEGFR-2 as a potential immuno- therapeutic target to mediate depletion of this highly suppressive cell subset, an implication that is supported by the finding that blockade of either VEGF-A or VEGFR-2 decreases intratumoral T regulatory populations and thereby enhances antitumor immunotherapies.140,143 Collectively, these studies support the hypothesis that tumor-derived VEGFs may contribute significantly to the generation of immunotolerant systemic and tumor (micro)environments.
Natural killer (NK) cells are a key cell subset involved in innate antitumor immunity. Interestingly, a recent study has identified VEGF-C as a key regulator of NK cell cytotoxicity and highlighted a critical role for this regulation in mediating immune tolerance.144 Noncytotoxic uterine NK cells were found to express significantly higher levels of VEGF-C when compared to their cytotoxic NK cell counterparts (both peripheral and uterine), which retained their cytotoxic activity. Given that both noncytotoxic and cytotoxic NK cells exhibited similar cytotoxic machinery profiles, VEGF-C–mediated effects on target cells were investigated. Cytoprotection by VEGF-C was primarily related to upregulation of target cell expression of TAP-1, a key molecule involved in MHC class I (MHC-CI) assembly.145 Indeed, MHC-CI molecule expression on target cells signals to suppress NK cytoxicity, while a lack of MHC-CI receptor engagement results in NK cell–mediated killing.146 Importantly, exogenous VEGF-C could rescue target cells from traditionally cytotoxic NK cells. Given that many parallels (particularly in relation to immune privilege) exist between the microenvironment established during normal human pregnancy and malignant progression,147 it is tempting to speculate that VEGF-C–mediated protection of target cells from cytotoxic NK cells may also play a role in immune suppression within the tumor microenvironment.
In addition to lymphangiogenic factors directly modulating the maturation, phenotype, activity, and recruitment of immune cell subsets, emerging evidence suggests that cells of the lymphatic vasculature may also play a role in sculpting host immunity to tumors, in particular mediating tumor tolerance.
Recently, our understanding of the mechanisms that govern peripheral tolerance of self-antigens has taken a significant shift from the previously accepted dogma.148 Autoreactive T cells that have escaped negative selection in the thymus are subject to mechanisms of peripheral tolerance. Their fate is determined in secondary lymphoid organs, where signaling from antigen-presenting cells, such as dendritic cells, in the absence of costimulatory signaling, results in either their deletion or the development of an anergic cell phenotype. Until recently, the accepted model of peripheral tolerance has relied heavily upon dendritic cells as the central cellular mediator of antigen presentation. Recent evidence, however, suggests a role for the lymphatic stroma in antigen presentation and subsequent clonal deletion of self-reactive T cells. Indeed, lymph node–resident lymphatic endothelial cells were found to express endogenous peripheral tissue antigens via MHC class I molecules independently of the autoimmune regulator (Aire).149 Specifically, lymphatic endothelial cells in skin-draining peripheral lymph nodes were shown to directly present an epitope of the melanocyte-specific tyrosinase protein, to tyrosinase-specific CD8+ T cells, resulting in their clonal deletion and thereby demonstrating a novel mechanism mediating peripheral tolerance. Therefore, lymphatic vessels themselves are important mediators of peripheral tolerance, not only via their ability to transport self-antigens to the lymph node but also via their ability to directly present endogenous antigen to self-reactive T cells, the result of which is deletional peripheral tolerance.150 It is therefore tempting to speculate that lymphatic endothelial cell–mediated presentation of tumor antigens may play a role in the development of immune tolerance of tumors.
A recent study has indicated the chemokine CCL21 may be a key modulator of immunity within the tumor microenvironment.151 In addition to its chemotactic properties for CCR7-expressing antigen-presenting cells and malignancies, CCL21 also acts as a chemokine for lymphoid tissue inducer (LTi) cells and is thereby a key driver of lymphoid neogenesis.152 New lymphoid-like reticular stromal networks were observed in the peritumoral regions of CCL21-overexpressing B16-F10 melanoma cells and were associated with the generation of an immunotolerant tumor microenvironment characterized by the infiltration of T regulatory cells, myeloid-derived suppressor cells, and an immunosuppressive cytokine milieu (Fig. 3).151 Importantly, B16 cells with reduced CCL21 expression grew at a reduced rate compared to their high CCL21-expressing counterparts. These studies highlight the potential for the lymphatic stroma as a key modulator of tumor-immune responses.
These findings are supported by another recent study, whereby a T regulatory cell precursor population (CD4+foxp3–) has been identified and characterized in peripheral lymphoid organs.153 Using unmanipulated mice, this study demonstrated that these precursor cells are able to differentiate extrathymically to CD4+foxp3+ T regulatory cells with potent immunosuppressive capabilities. The possibility exists (and remains to be determined) that lymphoid-like structures induced by VEGFs may provide a stromal niche for these regulatory precursor cells to differentiate into Foxp3+ T regulatory cells. Tumor-associated lymphatic T regulatory cell differentiation and the potential for these cells in the suppression of tumor-targeting immunity remain to be demonstrated (Fig. 3).
The interplay between VEGFs, lymphatic vessels, and immune cell subsets is highly complex, and clearly, the interaction between the key factors and cellular components needs to be further investigated. Taken collectively, however, these studies support the emerging hypothesis that tumor-induced lymphangiogenesis may regulate host immunity. Neolymphoid structures induced by tumor-derived VEGFs may upregulate the expression of factors such as CCL21 in the lymphatic endothelium. This may, in turn, mediate a switch in the tumor microenvironment from one conducive to T cell–mediated cytotoxicity to one dominated by immunosuppressive cytokines and regulatory cells. Moreover, the lymphatic endothelium may itself present tumor-derived antigens to CD8+ T cells, as previously observed with endogenous peripheral tissue antigens in the lymph node, resulting in deletion of tumor-reactive lymphocytes.153 Thus, tumor-associated lymphatic vessels may sculpt a tumor microenvironment that is permissive to malignant progression and metastasis. Further investigation of the interplay between lymphangiogenesis, activated lymphatic endothelial cells, and tumor immunity will importantly define what is a highly complex relationship.
Increasing evidence from both experimental murine tumor model systems and clinical data indicates that tumor-associated lymphangiogenesis promotes metastasis and that this has prognostic importance for patients. It is now emerging that the processes that regulate lymphatic metastasis are significantly more complex than those originally proposed. It is emerging that lymphatic metastasis involves intricate regulation by multiple soluble factors and cell types that not only drive the active migration of tumor cells into lymphatic vessels and beyond but may also act to regulate the balance between host immunity and tolerance.
Distal lymphangiogenesis in the lymph node, even prior to metastasis, is also emerging as a key feature that may impact multiple processes that act in the promotion of metastatic dissemination. In addition to providing a structural niche for metastatic cells, the altered lymph node vasculature and resultant increases in lymphatic flow may also act to sculpt an immune response permissive to metastatic spread. Increased lymphatic flow as a result of tumor-derived VEGFs may increase the chemotactic gradient for chemokines such as CXCL12 while also driving increased expression of the chemokine CCL21. CCL21, via its ability to alter the lymphoid stroma, may switch the host immune response from one that is immunogenic to one characterized by immunosuppressive cells and cytokines and thereby tolerant of tumor progression (Fig. 3). Moreover, a CCL21-driven immunosuppressive microenvironment may be further compounded by the direct effects of VEGFs on immune cell function. Inadequate antigen presentation by dendritic cells, resulting in both decreased activation of tumor-specific cytotoxic T lymphocytes and an increased proportion of suppressive T cells,132,138 coupled to direct inhibition of T cell proliferation would all act to exacerbate the generation of a tumor-tolerant environment. Moreover, the recently identified role for the lymphatic endothelium itself in the presentation of peripheral antigens, potentially including tumor-derived antigens, may lead to the deletion of tumor-specific CD8+ effector cells (Fig. 3).150 This picture of lymphatic-mediated immune regulation of metastatic dissemination is not yet clearly defined. Moreover, the precise role that lymphatic vessels play in programming tumor immunity, and the subsequent effects this has on tumor progression and metastasis, remains undefined. It is however clear that the further characterization of these processes will not only extend our knowledge of the tumor microenvironment but also potentially identify novel cancer immunotherapeutic targets for the more effective treatment of advanced cancers.
Declaration of Conflicting Interests: The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.
Funding: This work was supported by the Swiss National Science Foundation [grant numbers 3100A0108207 and 31003A-130627]; Commission of the European Communities [grant number LSHCCT2005518178]; European Research Council [grant number LYVICAM]; and Oncosuisse and Krebsliga Zurich (to M.D.).