MINI REVIEW Tumor metastasis and the lymphatic vasculature

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1 Int. J. Cancer: 125, (2009) ' 2009 UICC MINI REVIEW Tumor metastasis and the lymphatic vasculature Jonathan P. Sleeman 1,2 * and Wilko Thiele 1,2 1 Medical Faculty Mannheim, University of Heidelberg, Heidelberg, Mannheim, Germany 2 Forschungszentrum Karlsruhe, Institut f ur Toxikologie und Genetik, Postfach 3640, Karlsruhe, Germany Tumor-associated lymphatic vessels act as a conduit by which disseminating tumor cells access regional lymph nodes and form metastases there. Lymph node metastasis is of major prognostic significance for many types of cancer, although lymph node metastases are themselves rarely life-threatening. These observations focus our attention on understanding how tumor cells interact with the lymphatic vasculature, and why this interaction is so significant for prognosis. Tumors interact with the lymphatic vasculature in a number of ways, including vessel co-option, chemotactic migration and invasion into lymphatic vessels and induction of lymphangiogenesis. Tumor-induced lymphangiogenesis both locally and in regional lymph nodes has been correlatively and functionally associated with metastasis formation and poor prognosis. The investigation of the molecular regulation of lymphangiogenesis has identified ways of interfering with prolymphangiogenic signaling. Blockade of tumor-induced lymphangiogenesis in preclinical models inhibits metastasis formation in lymph nodes and often also in other organs, suggesting that blocking the lymphatic route of dissemination might suppress metastasis formation not only in lymph nodes but also in other organs. However, randomized clinical trials that have investigated the efficacy of therapeutic removal of lymph nodes have concluded that lymph node metastases act only as indicators that primary tumors have developed metastatic potential, and do not govern the further spread of metastatic cells. To reconcile these apparently paradoxical observations we suggest a model in which tumor-induced lymphangiogenesis and lymph node metastasis formation act as indicators that tumors are producing factors that can act systemically to promote metastasis formation in distant organs. ' 2009 UICC Key words: lymphangiogenesis; lymphatic endothelial cells; lymphatic vessels; cancer; metastasis; lymph nodes; VEGFR-3; VEGF-C; VEGF-D Metastasis is thought to be responsible directly or indirectly for more than 90% of all cancer deaths. 1 Thus the prognosis of cancer patient deaths is intimately connected with the process of metastasis. Systemic metastases arise from tumor cells that disseminate via the blood and lymphatic vasculature. Lymph nodes are often the first organs in which metastases form. 2 They are rarely lethal in themselves, and in the main can usually be surgically removed without difficulty. 3 However, for many types of carcinoma, the presence of metastatic tumor cells in regional lymph nodes is one of the most important indicators of poor prognosis and forms the basis of the majority of tumor staging schemes. 4,5 Sentinel lymphonodectomy, a surgical technique in which the first or sentinel lymph node that drains a primary tumor is removed and analyzed for the presence of metastatic tumor cells, is now widely used to guide further therapy of postoperative cancer patients. 6 The importance of lymph node metastases in predicting the course of neoplastic disease focuses our attention on understanding how tumors interact with the lymphatic vasculature, the conduit that brings disseminating cells to regional lymph nodes, and what significance this has on determining the development of metastases in vital organs. Interactions between tumor cells and the lymphatic system that promote metastasis In recent years a variety of mechanisms have been uncovered that serve to support metastatic dissemination via the lymphatic vasculature. The architecture of lymphatic vessels is determined by their function in absorbing interstitial fluid and allowing immune cells to traffick, and makes them intrinsicly more amenable to the entry of invasive tumor cells in comparison to blood vessels. For example, they have loose overlapping cell cell junctions, and end capillaries have no or only an incomplete basement membrane. 7 Invasive tumors can permeate into the lymphatic vasculature locally as strings of cells, but generally traffick through the lymphatics as emboli. 8 Malignant tumors are often associated with an increased density of lymphatic vessels, mostly in the peritumoral region, but also to a lesser degree intratumorally. This may be caused by cooption of pre-exisiting lymphatic vessels by the growing tumor, or by induction of lymphangiogenesis, the outgrowth of new lymphatic vessels. Assessment of the proliferative index in tumor-associated lymphatic endothelial cells (LECs) suggests that both mechanisms can be operative. 8 Intratumoral lymphatic vessels are considered to be rare 9 and probably nonfunctional due to mechanical compression, 10 although the frequency and functionality of intratumoral lymphatic vessels remains a topic of debate. Increased lymphatic vessel density (LVD) in and around tumors is thought to increase the chance that invasive tumor cells enter the lymphatic vasculature. In turn, it is suggested that this promotes the formation of lymph node metastases, as increased numbers of disseminating tumor cells are transported to regional lymph nodes. The induction of lymphangiogenesis by tumors is mediated by prolymphangiogenic growth factors and cytokines that can be produced by the tumor cells themselves, or by stromal cells, tumorassociated macrophages or damaged platelets The most widely studied factors in this context are members of the VEGF family and their cognate receptors VEGFR-2 and VEGFR-3. Recent studies show that in addition to inducing local lymphangiogenesis, tumor-derived VEGF-A and VEGF-C can promote lymphangiogenesis systemically within tumor-draining lymph nodes. 15,16 This can even occur in the absence of peri-tumoral lymphangiogenesis. 17,18 Remote induction of intranodal lymphangiogenesis by tumors is suggested to promote the flow of lymphatic fluid from primary tumors to lymph nodes, increasing the efficacy of transport of disseminating tumor cells to the lymph nodes and thereby fostering the formation of lymph nodes metastases. 17 Chemokines also play a role in promoting the entry of tumor cells into the lymphatics. LECs have been shown to secrete CCL Tumor cells that express the cognate receptor for this Grant sponsor: European Union (FP7 collaborative project TuMIC); Grant number: HEALTH-F ; Grant sponsor: Deutsche Forschungsgemeinschaft [Schwerpunktprogramm SPP1190 (Tumor-vessel interface)]. *Correspondence to: Centre for Biomedicine and Medical Technology Mannheim (CBTM), Universit atsmedizin Mannheim, University of Heidelberg, TRIDOMUS-Geb aude Haus C, Ludolf-Krehl-Str , D Mannheim, Germany. Fax: sleeman@medma.uni-heidelberg.de Received 25 March 2009; Accepted after revision 22 June 2009 DOI /ijc Published online 30 June 2009 in Wiley InterScience ( wiley.com). Publication of the International Union Against Cancer

2 2748 SLEEMAN AND THIELE chemokine, CCR-7, have been shown to chemotactically migrate towards CCL21-producing LECs both in vitro and in vivo. 20 Furthermore, autologous chemotaxis mechanisms have been uncovered by which disseminating tumor cells detect and migrate with the flow of interstitial fluid, thereby promoting their entry into the lymphatic vasculature. For example, certain CCR-7-postive tumor cells have been shown to express CCL-21, thereby setting up an autocrine stimulatory loop. Flow of interstitial fluid around the tumor cells washes chemokine away from the windward side of the cell, but not so effectively from the leeward side. This creates a chemokine gradient across the cell to which it can respond and migrate in the direction of fluid flow towards the draining lymphatics. 21 VEGF-C has also been reported to play an analogous role in promoting invasion into the lymphatics of VEGFR-3-positive tumor cells by both autocrine and CCR7-dependent paracrine signaling mechanisms, 22 although the generality of the expression of VEGFR-3 on carcinoma cells is controversial. 23,24 Recent studies suggest that tumors can induce immune suppression in regional lymph nodes, as evidenced by tumor-induced changes in the immune cell populations found in sentinel lymph nodes of melanoma and breast cancer patients These changes include reduced numbers of high endothelial venules, dendritic cells and CD41 and CD81 T-cells, anergy of CD81 T-cells 30 and increased levels of immunosuppressive cytokines in sentinel lymph nodes. 31 The relevance of these changes for lymph node metastasis formation remains unclear, but they seem to be relevant for patient survival. 29 Lymphangiogenesis and metastasis: Relevance and function Since the advent of the molecular age of lymphangiogenesis research following the discovery of the archetypal lymphangiogenesis regulator VEGFR-3, it has become well established that many different types of carcinoma are able to induce lymphangiogenesis. To assess the significance of lymphangiogenesis for tumor metastasis, it is important to address how frequently lymphangiogenesis is induced by tumors, what its significance is for prognosis, and what its functional relevance is to disease outcome. Answers to these questions come from correlative studies on human tumor material and loss and gain of function experiments in animal tumor models. At the time of writing this article, more than 700 articles have been published concerning cancer and lymphangiogenesis. Most of these have focused on the staining of sections from human tumors for lymphatic vessels or pro-lymphangiogenic growth factors such as VEGF-C and -D, the known ligands for VEGFR-3, and then have correlated these findings with a variety of clinical parameters. Specific staining for lymphatic vessels has been facilitated by the discovery of several proteins whose preferential expression in LECs allows them to be used as markers. These include VEGFR-3, the mucin-type transmembrane protein, 32 the LEC hyaluronan receptor LYVE-1, 33 and the homeobox transcription factor Prox As none of these markers are exclusively or homogeneously expressed on tumor lymphatics, 8 a combination of markers is needed to identify tumor-associated lymphatic vessels in a reliable manner. Although combinations of markers have not always been strictly employed in published studies using human tumor material, important conclusions concerning tumor-associated lymphatics can nevertheless be gleaned from the extensive literature. Table I summarizes the outcome of published studies that attempt to correlate LVD with lymph node metastasis formation and patient survival. This table demonstrates that an enhanced density of lymphatic vessels both peritumorally and/or intratumorally is associated with lymph node metastasis formation and poor prognosis for some but not all types of cancer. For several types of cancer, conflicting data have been published, with some studies reporting a correlation, others not. Remote induction of lymphangiogenesis by tumors in draining lymph nodes has also been reported, for example in human breast tumors, where lymphangiogenesis was observed in 25% of noninvolved axillary lymph nodes 88 Several factors probably account for the lack of tight correlation between LVD, lymph node metastasis and patient prognosis. Co-option of preexisting lymphatic vessels by tumors within tissues having high local LVD may suffice to support lymphogenic spread of tumor cells in the absence of lymphangiogenesis. Indeed, significant numbers of studies have reported no increased proliferation of LECs in tumor-associated lymphatics. 8 Proliferating LECs are also not the only cells that can contribute to the formation of new lymphatic vessels. Bone marrow-derived endothelial precursor cells and CD11b1 macrophages can both contribute to the lymphatic vasculature in tumors, although the degree to which these cells contribute varies widely in different reports The apparent detection of intratumoral vessels may reflect the involvement of macrophages, as LYVE-1 is expressed on CD11b1 macrophages in the context of tumors and is often used as a marker for detecting intratumoral lymphatics. 91 CD11b1LYVE-11 macrophages may contribute to lymphatic vessels through vascular mimicry or transdifferentiation. 91,93,94 In animal models, inhibition of the activity of VEGF-A, VEGF- C, VEGF-D, COX-2 and PDGF-BB in the context of tumors has been shown to suppress tumor-induced lymphangiogenesis, indicating that these factors have the capacity to contribute to tumor-induced lymphangiogenesis. COX-2 is likely to act indirectly by inducing expression of VEGF-C in macrophages. 97 These observations are relevant to human disease, as studies with human tumor samples show that a variety of different types of cancer express VEGF-C and/or VEGF-D. 100,101 In many of these studies the expression of these growth factors in tumors was found to correlate with LVD, lymph node metastasis and poor prognosis. However, this was not always the case, indicating that there is not necessarily a direct link between the expression of VEGF-C and VEGF-D, LVD and metastasis formation. 100 Ectopic overexpression of VEGF-C or VEGF-D in tumor cells that can be grown as tumors in vivo, or transgenic expression of these factors in organ compartments prone to the development of tumors has been shown to promote lymphangiogenesis in variety of animal tumor models. Enhanced proliferation rates in LECs of tumor-associated lymphatic vessels and increased LVD and/or lymphatic vessel diameter are thereby induced. Concomitantly, an increased incidence of metastasis formation in regional lymph nodes has been observed, and in many studies also in vital organs such as the lung. 16,95, In complementary approaches, ligand-induced activation of VEGFR-3 has been suppressed in several different animal tumor models, for example through the ectopic expression of soluble VEGFR-3 fusion proteins. Tumorinduced lymphangiogenesis was thereby inhibited but there was no effect on preexisting vessels. Most significantly, the onset or incidence of lymph node metastases was retarded or reduced, and in many cases the formation of metastases in other organs such as the lung was also inhibited. 56,95,105, These data have been used to support the idea that activation of VEGFR-3 signaling in LECs by tumor-derived VEGF-C and -D promotes lymphangiogenesis in the vicinity of the tumor, thereby increasing the likelihood that invasive tumor cells will enter the lymphatic vasculature and traffick to regional lymph nodes and beyond. Taken together, the large body of evidence from correlative studies using human tumor samples and functional studies with animal models indicate that tumor-induced lymphangiogenesis does occur in some but not all tumors. Where it does occur, it is often associated with metastasis, particularly within regional lymph nodes but also in other organs. However, tumor-induced lymphangiogenesis is not an obligatory feature of tumor progression, and metastasis formation can occur in its absence. This reflects complex relationships between tumors and lymphatic vessels that are different not only for different types of cancer, but also for each individual tumor depending on its precise location and genetic constitution.

3 METASTASIS AND THE LYMPHATICS 2749 TABLE I SUMMARY OF THE LITERATURE THAT HAS ADDRESSED THE CORRELATION BETWEEN LYMPHATIC VESSEL DENSITY IN DIFFERENT TYPES OF CANCER AND LYMPH NODE METASTASIS FORMATION (LN MET.) AND/OR POOR PROGNOSIS (SURVIVAL) Tumor type LN met. Survival Marker References 35 Breast /CD /CD31/CD34 Bladder tc Podopanin Cervical (Colo)rectal /vwf Endometrial Gastric Hilar cholangio 1 1 HNSCC /CD Melanoma / /CD /CD31 64 Mesothelioma nucleotidase 65 Nsc lung 1 VEGFR-3/CD /Podolpanin/CD VEGFR-3//CD Oesophageal Oral scc 1 2 /CD Ovary 2 Pancreatic / Prostate 2 /CD /VEGFR-3/CD Thyroid Renal cell /CD34 Upper tract urothelial /CD34 The markers used to assess lymphatic vessel density are indicated. Correlation, 1; no correlation, 2. 1 but correlation between intratumoral lymphatic vessel density inside the lymph node metastases and patient survival. 2 significant association of LVD with LN mets and survival, but LVD was not an independent factor for poor prognosis in multivariate analysis. 3 only peritumoral but not intratumoral LVD. 4 neither peritumorally nor intratumorally. 5 peritumoural LVD, but not intratumoural, correlated with liver metastasis. 6 only intratumoral but not peritumoral LVD. 7 both peritumoral and intratumoral LVD. 8 association of both peritumoral and intratumoral LVD with more rapid development of lymph node metastasis. 9 only in angiogenic tumors and only for peritumoral but not intratumoral LVD. 10 only intratumoral LVD was assessed. 11 only intratumoral LVD was assessed; no influence on overall survival in univariate analysis, but significant association between IL and disease-free survival. 12 but correlation with liver metastases. Signaling lymphangiogenesis: Maintenance and induction There has been a dramatic increase of our understanding of the molecular regulation of lymphangiogenesis in recent years. Several of the pathways involved have been demonstrated to be operative during tumor-induced lymphangiogenesis. In the following section we summarize our current understanding of how lymphangiogenesis is regulated at the molecular level (see also Fig. 1). The homeobox transcription factor Prox1 is a master regulator of LEC identity, and its continued expression is vital for lymphatic endothelial cells to maintain their identity. 110 During embryogenesis,

4 2750 SLEEMAN AND THIELE FIGURE 1 Schematic diagram demonstrating the interconnection between major molecular pathways that regulate lymphatic endothelial cell identity (left side) and lymphangiogenesis (right side). The interaction between extracellular and cell surface regulators with cytoplasmic mediators (encompassed by the green box) and nuclear transcription factors (encompassed by the yellow semi-circle) is illustrated. Full details and references are to be found within the main text. induction of Prox1 expression in anterior cardinal vein endothelial cells at E9 by Sox18 is the earliest known molecular event in the specification of the lymphatic vasculature. 111 Positive feedback loops have been described that serve to maintain Prox1 expression in LECs. For example, Prox1 induces expression of IL-3, which then feeds back onto LECs to maintain expression of Prox Another interleukin, IL-7, has also been implicated in promoting Prox-1 expression. 113 In addition to Sox18 and Prox1, other transcription factors such as Foxc2 and Net/Elk3, have been implicated in determining LEC identity. 114 Prox1 expression drives the transcription of a variety of genes whose expression is associated with key LEC characteristics. 115,116 Some of these genes themselves play decisive roles in determining LEC identity, such as podoplanin. Loss of podoplanin or suppression of its O-glycanation disrupts LEC identity. 117,118 Other Prox1-regulated genes include a9b1 integrin and the transmembrane receptor tyrosine kinases VEGFR-3 and FGFR-3 that play important roles in regulating lymphangiogenesis. 115,116,119,120 At least part of this transcriptional regulation is dependent on physical interaction between Prox1 and the orphan nuclear receptor COUP-TFII. 121,122 Thus a prerequisite for lymphangiogenesis is expression of Prox1, as it maintains LEC identity and ensures that the molecular machinery required to transduce prolymphangiogenesis signals is present. The transcription factor NFATc1 may play a similar role, as its expression has recently been shown to be required for FGFR-3, VEGFR-3 and podoplanin expression in LECs. 123 The importance of maintaining LEC characteristics for lymphangiogenic signaling has recently been underscored by the finding that TGF-b signaling acts as a negative regulator of lymphangiogenesis and suppresses the expression of genes that determine LEC characteristics. 124 The most intensively studied molecular mechanism that induces lymphangiogenesis is the activation of VEGFR-3 by VEGF-C and VEGF-D. 114 Ligand-induced autophosphorylation of VEGFR-3 activates several signal transduction pathways that in turn regulate expression of a variety of genes. 101 Co-receptors play an important role in regulating ligand-induced VEGFR-3 activation. The a9b1 integrin binds to VEGF-A, -C and -D, and the lymphangiogenesisstimulating activity of VEGF-C and -D has been shown to be dependent on a9b1 integrin. 125 Neuropilin-2 binds to VEGF-C and -D, is co-internalized with VEGFR-3 upon ligand binding and is thought to regulate VEGFR-3 activation. 126 The central role of VEGFR-3 activation in driving lymphangiogenesis is reflected in its prominent expression on the tip cells of sprouting lymphatic capillaries that are crucial for the outgrowth of new lymphatic vessels. 127 Proteolytic processing of VEGF-C and VEGF-D allows these growth factors to bind to and activate VEGFR-2; another member of the vascular endothelial growth factor receptor family that is expressed on LECs, and which has also been implicated in the regulation of lymphangiogenesis Activation of VEGFR-2 by its archetypal ligand VEGF-A has been reported to induce lymphatic hyperplasia. 15,131 Interestingly, activation of VEGFR-2 and VEGFR-3 expressed on LECs has distinct effects on the lymphatic vasculature. VEGFR-2 activation induces vessel enlargement, while VEGFR-3 activation leads to sprouting lymphangiogenesis. 127 In addition, molecular interactions between VEGFR-2 and VEGFR-3 may be decisive for the cellular response to ligand, as VEGF-D has been shown to induce the formation of heterodimers between VEGFR-2 and VEGFR-3, potentially leading to differences in the signal transduction pathways that are subsequently activated. 132,133 The picture that is emerging is that VEGFR-2 and VEGFR-3 co-operate to regulate LEC migration and proliferation 134 and that VEGFR-2 activation is a modifier but not necessarily an initiator of lymphangiogenesis. 127 In addition to VEGFR-2 and-3, a number of other cell surface receptors can induce lymphangiogenesis in response to their cognate ligands. These include the receptor tyrosine kinases Tie-1 and Tie-2 and their ligands angiopoietin-1 (Ang-1) and Ang-2, 135 the hepatocyte growth factor receptor c-met, 136 EphrinB2, 137 and receptors for platelet-derived growth factor, 96 lymphotoxin beta, 138 insulin-like growth factors 1 and 2 and members of the fibroblast growth factor family. 117,139,140 Some of these receptor ligand interactions indirectly promote lymphangiogenesis by inducing the expression of prolymphangiogenic factors or by upregulating the expression of the receptors for prolymphangiogenic factors. Integrins other than a9b1 have also been associated with lymphangiogenesis. For example, a1b1, a2b1 and a5b1 have been implicated in regulating lymphangiogenesis in the context of wound healing and inflammation. 141,142 Furthermore, the extracellular matrix elastic microfibril-associated protein EMILIN1 is a ligand for a4b1, 143 and EMILIN deficiency has pronounced effects on lymphatic vessel growth and function, 144 potentially implicating a4b1 in lymphangiogenesis. To date, only VEGFR-2, VEGFR-3 and PDGFR activation has been shown to play a role tumor-induced lymphangiogenesis. Other known prolymphangiogenic receptor-ligand pairs may also be involved in the context of tumors, but this remains to be demonstrated. Lymphangiogenesis describes the co-ordinated regulation of several cellular processes involving LEC proliferation, migration, invasion and tubule formation. While growth factors and their

5 METASTASIS AND THE LYMPHATICS 2751 receptors that induce lymphangiogenesis are comparatively well described, the intracellular signal transduction pathways and transcription factors that ultimately co-ordinate the complex lymphangiogenic process are less well understood. Protein kinase C-dependent activation of the MAPK signaling cascade (ERK, JNK) and induction of Akt phosphorylation seem to play central roles. 133,145 Thus, activation of VEGFR-3 by its ligands VEGF-C or VEGF-D results in ligand-induced phosphorylation of VEGFR- 3 tyrosine residues 1,063 and 1,230/1,231 on the VEGFR-3 cytoplasmic tail. The former phosphorylation recruits CRKI/II which in turn induces expression of the transcription factor c-jun via JNK1/2, while the latter recruits GRB2, subsequently activating ERK1/2 and AKT. 145 Physiological counter-regulation of these signaling pathways is provided by members of the sprouty/spred family of proteins that can inhibit prolymphangiogenic VEGF-C signaling by suppressing VEGFR-3-mediated ERK and Akt activation. 146 Lymphangiogenic signaling by FGF-2 also activates the Akt/mTOR/p70S6 kinase pathway, 147 possibly indicating convergence of prolymphangiogenesis signaling pathways on Akt. Consistently, a specific inhibitor of mtor called rapamycin is able to inhibit tumor-induced lymphangiogenesis and lymphatic metastasis. 145 However, it is also important to bear in mind that FGF-2 upregulates VEGF-C expression, and that VEGF-C expression is suppressed by rapamycin, 148,149 and thus alternative interpretations of these data are possible. The connection between signal transduction pathways activated by prolymphangiogenic factors and the transcriptional machinery largely remains to be defined. However, gene expression profiling has revealed panels of genes that are regulated by VEGF-A and VEGF-C in LECs. 150 One of the most highly upregulated genes was ESM1, a secreted glycoprotein that is specifically expressed in LECs and which potentiates their proliferation and migration. Translation and clinical contradictions There are several scenarios in which suppression of metastasis formation in cancer patients could have therapeutic application. 151 There has therefore been much interest in the possibility that the tumor lymphatics could serve as a target for developing new therapeutic strategies for cancer, particularly because of the (at least partial) correlation between the expression of prolymphatic growth factors and LVD on one hand, and lymph node metastasis formation and poor prognosis on the other, as well as functional studies in animal models that provide evidence that inhibition of tumor-induced lymphangiogenesis can suppress metastasis. In preclinical studies, a variety of approaches have been taken to suppress tumor-induced lymphangiogenesis, virtually all of which target components of signal transduction cascades that orchestrate lymphangiogenesis outlined above. The approaches include antibodies that target either prolymphangiogenic growth factors or their receptors to prevent activation of the receptor, soluble receptors to sequester and neutralize prolymphangiogenic growth factors, synthetic chemical inhibitors and naturally-occurring substances that block activation of receptors or other signal transduction components, and shrna to inhibit expression of key genes that are transcribed in response to prolymphangiogenic signaling. 8 Although preclinical studies demonstrate the utility of a variety of methods for blocking tumor-induced lymphangogienesis, there remain a number of issues to be resolved regarding the therapeutic targeting of tumor-associated lymphatic vessels. As multiple growth factors are probably involved in regulating tumor-induced lymphangiogenesis, blocking only one or even several of these factors may not effectively suppress tumor-induced lymphangiogenesis. 152 Furthermore, as outlined above it is clear from studies on human cancers that tumor-induced lymphangiogenesis does not always contribute to the LVD in the vicinity of tumors. Inhibition of lymphangiogenesis does not seem to affect preexisting vessels, 153 and vessels induced in the context of corneal lymphangiogenesis assays or chronic inflammation do not regress after withdrawal of the original prolymphangiogenic stimulus. 139,154 Thus, if tumors have already induced lymphangiogensis, or for those tumors where vessel co-option is the major source of tumor-associated lymphatics, inhibition of lymphangiogenesis is unlikely to be effective. Possible unwanted side effects of targeting tumorassociated lymphatic vessels also have to be considered. 100 Lymphangiogenesis is induced after wounding 155 and so inhibition of lymphangiogenesis might interfere with wound healing and tissue regeneration. Other potential side effects will be dependent on the molecular pathway that is targeted. In addition to these considerations, there is a much more central issue to be resolved that goes to the heart of our understanding of the process of metastasis. Lymph node metastases are rarely life threatening. 3 If targeting the tumor lymphatics is to have a significant impact on prognosis, then tumor-induced lymphangiogenesis needs to play a central role in determining the development of metastasis not only in lymph nodes but also more widely in vital organs. In turn this raises the question of the degree to which lymph node metastases and the trafficking of disseminating tumor cells via the lymphatics are involved in governing the formation of metastases in other organs. In other words, to what extent are metastases that develop in vital organs such as the lung derived from tumor cells that have disseminated via the lymphatics? It is clear from the literature the tumor cells traffick via both blood and lymphatic vessels in an interrelated manner. 8 However, only in the case that tumor cells that disseminate through the lymphatics make a significant contribution to metastasis in vital organs will therapies directed against tumor lymphatics be effective. The predominant prognostic value of lymph node metastases in predicting disease outcome, together with the now established value of sentinel lymphonodectomy in the clinical management of cancer would seem to speak for a role in lymph node metastases and thus trafficking of tumor cells via the lymphatic route in determining patient prognosis which generally means the development of metastases in vital organs. This notion is supported by the observations outlined above regarding the correlation that often exists between expression of prolymphangiogenic factors and LVD on one hand, and lymph node metastasis and poor prognosis on the other. Furthermore, animal experiments in which tumorinduced lymphangiogenesis is experimentally modulated again seem to attest to a major functional role for the lymphatic route of dissemination as not only is the formation of lymph node metastases influenced, but also in other organs such as the lung. The notion that lymph node metastases are essential way stations along the road towards the formation of metastases in vital organs has a long history. 8 According to this idea, removal of regional lymph nodes containing metastatic cells should have important survival advantages for cancer patients, as a key step in the metastatic cascade would be removed and further metastatic spread should be inhibited. This idea was the raison d être for a number of surgical procedures developed in the last century, most notably the mastectomy protocol of Halstead. 156 However, it was not until long term randomized clinical trials were initiated in the 1960s that the efficacy of these procedures in terms of improving cancer patient survival was examined. The startling result from several such clinical trials, particularly for melanoma and breast cancer, is that it makes very little difference to patient survival if regional lymph nodes are left in situ after removal of the primary tumor This is despite the fact that in a significant proportion of cases regional lymph node metastases subsequently developed and needed to be removed surgically. In these studies, lymph node metastasis formation was nevertheless found to be a strong indicator of poor prognosis, consistent with established grading schemes. The conclusion from these long-term studies where large cohorts of patients have been followed up for several decades is that lymph node metastases do not govern poor prognosis and thus distant metastases but merely act as indicators that the tumors from which they are derived have developed the potential to form metastases not only in regional lymph nodes but also in distant organs.

6 2752 SLEEMAN AND THIELE Perspective: Is tumor-induced lymphangiogenesis only an indicator and not a governor? It is clear that there is contradiction between clinical studies that address the role of lymph node metastases for patient survival, and our current interpretation of correlative and functional studies that have examined the significance of tumor-induced lymphangiogenesis for metastatic spread. How might this apparent paradox be resolved? One hypothesis would be that prolymphangiogenic factors act not only locally to induce tumor-associated lymphangiogenesis and lymph node lymphangiogenesis, but also systemically to promote metastasis in vital organs in ways that may be distinct from their prolymphangiogenic activity. While it must be stressed that such a scenario largely remains hypothetical and speculative, several observations provide initial support for this notion. As pointed out above, prolymphangiogenic factors act not only locally, but also systemically within the lymphatic system to induce lymphangiogenesis in the lymph nodes ,88 Whether these factors also induce lymphangiogenesis systemically in other organs remains to be examined. Nevertheless, it is clear from studies that have examined the levels of prolymphangiogenic growth factors in the blood of cancer patients that many patients exhibit a significant increase in these factors, and that this increase significantly correlates with poor prognosis. 53, Thus, systemically increased levels of these factors may be able to act to promote metastasis. In this regard it is interesting to note that VEGF-A, a factor involved in the regulation of tumor-induced lymphangiogenesis, is also able to mobilize bone marrow-derived cells. 164 Mobilization of bone marrow-derived cells is thought to be an important step in the creation of niche structures that support metastatic growth. 165 This could be one way in which growth factors that induce tumor-induced lymphangiogenesis locally can also promote metastasis in vital organs systemically. Thus one and the same tumor-derived growth factor may promote metastasis in 2 independent ways: local lymphangiogenesis in the vicinity of the primary tumor and the formation of lymph node metastases on the one hand, and systemic induction of metastasis formation in vital organs operative by an independent molecular activity of the growth factor on the other (Fig. 2). In this scenario, tumorinduced lymphangiogenesis and lymph node metastasis formation would have prognostic significance because they would act as indicators that tumors are producing factors that systemically promote metastasis formation in vital organs. However, they FIGURE 2 Hypothetical model in which prolymphangiogenic factors produced by tumors have both local and systemic effects. These factors induce lymphangiogenesis locally and promote metastasis to lymph nodes. The same factors produced systemically act in distant vital organs to promote metastasis formation, possibly by mechanisms that are distinct from their prolymphangiogenic activity. themselves would not take part in the inductive events in these vital organs, and therefore removal of the lymph node metastasis would not have an overt effect on patient survival. As a corollary, therapeutic targeting the activity of these prolymphangiogenic factors might inhibit metastasis formation, but not necessarily as a consequence of suppressing tumor-induced lymphangiogenesis. In conclusion, although great strides have been made over the last 15 years in understanding the molecular regulation of tumorinduced lymphangiogenesis and its relevance to metastasis development, additional studies are required to resolve currently paradoxical findings regarding the significance of tumor lymphatics and lymph node metastases for patient prognosis and the development of metastases in vital organs. The outcome of these studies will impact greatly on our understanding of the fundamental process of metastases, and will be decisive in revealing the extent to which tumor-associated lymphatics really represent a valid translational target for cancer therapy. 1. Sporn MB. The war on cancer. Lancet 1996;347: Sleeman JP. The lymph node as a bridgehead in the metastatic dissemination of tumors. Recent Results Cancer Res 2000;157: Cady B. Regional lymph node metastases: a singular manifestation of the process of clinical metastases in cancer: contemporary animal research and clinical reports suggest unifying concepts. Ann Surg Oncol 2007;14: Beahrs O, Myers M. Purpose and principles of staging. In: Beahrs O, Myers M, editors. Manual for staging of cancer. Philadelphia: Lippincott, Leong SP, Cady B, Jablons DM, Garcia-Aguilar J, Reintgen D, Jakub J, Pendas S, Duhaime L, Cassell R, Gardner M, Giuliano R, Archie V, et al. Clinical patterns of metastasis. Cancer Metastasis Rev 2006; 25: Tanis PJ, Nieweg OE, Valdes Olmos RA, Rutgers EJ Th, Kroon BB. History of sentinel node and validation of the technique. Breast Cancer Res 2001;3: Sleeman JP, Krishnan J, Kirkin V, Baumann P. Markers for the lymphatic endothelium: in search of the holy grail? Microsc Res Tech 2001;55: Sleeman JP, Schmid A, Thiele W. Tumor lymphatics. Semin Cancer Biol 2009; doi: /j.semcancer Weiss L. Metastasis of cancer: a conceptual history from antiquity to the 1990s. Cancer Metastasis Rev 2000;19: Leu AJ, Berk DA, Lymboussaki A, Alitalo K, Jain RK. Absence of functional lymphatics within a murine sarcoma: a molecular and functional evaluation. Cancer Res 2000;60: References 11. Wartiovaara U, Salven P, Mikkola H, Lassila R, Kaukonen J, Joukov V, Orpana A, Ristimaki A, Heikinheimo M, Joensuu H, Alitalo K, Palotie A. Peripheral blood platelets express VEGF-C and VEGF which are released during platelet activation. Thromb Haemost 1998; 80: Trikha M, Nakada MT. Platelets and cancer: implications for antiangiogenic therapy. Semin Thromb Hemost 2002;28: Schoppmann SF, Birner P, Stockl J, Kalt R, Ullrich R, Caucig C, Kriehuber E, Nagy K, Alitalo K, Kerjaschki D. Tumor-associated macrophages express lymphatic endothelial growth factors and are related to peritumoral lymphangiogenesis. Am J Pathol 2002; 161: Neuchrist C, Erovic BM, Handisurya A, Fischer MB, Steiner GE, Hollemann D, Gedlicka C, Saaristo A, Burian M. Vascular endothelial growth factor C and vascular endothelial growth factor receptor 3 expression in squamous cell carcinomas of the head and neck. Head Neck 2003;25: Hirakawa S, Kodama S, Kunstfeld R, Kajiya K, Brown LF, Detmar M. VEGF-A induces tumor and sentinel lymph node lymphangiogenesis and promotes lymphatic metastasis. J Exp Med 2005;201: Hirakawa S, Brown LF, Kodama S, Paavonen K, Alitalo K, Detmar M. VEGF-C-induced lymphangiogenesis in sentinel lymph nodes promotes tumor metastasis to distant sites. Blood 2007;109: Harrell MI, Iritani BM, Ruddell A. Tumor-induced sentinel lymph node lymphangiogenesis and increased lymph flow precede melanoma metastasis. Am J Pathol 2007;170:

7 METASTASIS AND THE LYMPHATICS Ruddell A, Kelly-Spratt KS, Furuya M, Parghi SS, Kemp CJ. p19/arf and p53 suppress sentinel lymph node lymphangiogenesis and carcinoma metastasis. Oncogene 2008;27: Mashino K, Sadanaga N, Yamaguchi H, Tanaka F, Ohta M, Shibuta K, Inoue H, Mori M. Expression of chemokine receptor CCR7 is associated with lymph node metastasis of gastric carcinoma. Cancer Res 2002;62: Shields JD, Emmett MS, Dunn DB, Joory KD, Sage LM, Rigby H, Mortimer PS, Orlando A, Levick JR, Bates DO. Chemokine-mediated migration of melanoma cells towards lymphatics a mechanism contributing to metastasis. Oncogene 2007;26: Shields JD, Fleury ME, Yong C, Tomei AA, Randolph GJ, Swartz MA. Autologous chemotaxis as a mechanism of tumor cell homing to lymphatics via interstitial flow and autocrine CCR7 signaling. Cancer Cell 2007;11: Issa A, Le TX, Shoushtari AN, Shields JD, Swartz MA. Vascular endothelial growth factor-c and C-C chemokine receptor 7 in tumor cell-lymphatic cross-talk promote invasive phenotype. Cancer Res 2009;69: Laakkonen P, Waltari M, Holopainen T, Takahashi T, Pytowski B, Steiner P, Hicklin D, Persaud K, Tonra JR, Witte L, Alitalo K. Vascular endothelial growth factor receptor 3 is involved in tumor angiogenesis and growth. Cancer Res 2007;67: Petrova TV, Bono P, Holnthoner W, Chesnes J, Pytowski B, Sihto H, Laakkonen P, Heikkil a P, Joensuu H, Alitalo K. VEGFR-3 expression is restricted to blood and lymphatic vessels in solid tumors. Cancer Cell 2008;13: Cochran AJ, Wen DR, Farzad Z, Stene MA, McBride W, Lana AM, Hoon DS, Morton DL. Immunosuppression by melanoma cells as a factor in the generation of metastatic disease. Anticancer Res 1989; 9: Reiss CK, Volenec FJ, Humphrey M, Singla O, Humphrey LJ. The role of the regional lymph node in breast cancer: a comparison between nodal and systemic reactivity. J Surg Oncol 1983;22: Cochran AJ, Huang RR, Lee J, Itakura E, Leong SP, Essner R. Tumour-induced immune modulation of sentinel lymph nodes. Nat Rev Immunol 2006;6: Huang RR, Wen DR, Guo J, Giuliano AE, Nguyen M, Offodile R, Stern S, Turner R, Cochran AJ. Selective modulation of paracortical dendritic cells and T-lymphocytes in breast cancer sentinel lymph nodes. Breast J 2000;6: Kohrt HE, Nouri N, Nowels K, Johnson D, Holmes S, Lee PP. Profile of immune cells in axillary lymph nodes predicts disease-free survival in breast cancer. PLoS Med 2005;2:e Preynat-Seauve O, Contassot E, Schuler P, Piguet V, French LE, Huard B. Extralymphatic tumors prepare draining lymph nodes to invasion via a T-cell cross-tolerance process. Cancer Res 2007; 67: Kim R, Emi M, Tanabe K, Arihiro K. Immunobiology of the sentinel lymph node and its potential role for antitumour immunity. Lancet Oncol 2006;7: Breiteneder-Geleff S, Soleiman A, Kowalski H, Horvat R, Amann G, Kriehuber E, Diem K, Weninger W, Tschachler E, Alitalo K, Kerjaschki D. Angiosarcomas express mixed endothelial phenotypes of blood and lymphatic capillaries: podoplanin as a specific marker for lymphatic endothelium. Am J Pathol 1999;154: Banerji S, Ni J, Wang SX, Clasper S, Su J, Tammi R, Jones M, Jackson DG. LYVE-1, a new homologue of the CD44 glycoprotein, is a lymph-specific receptor for hyaluronan. J Cell Biol 1999;144: Wigle JT, Oliver G. Prox1 function is required for the development of the murine lymphatic system. Cell 1999;98: Bono P, Wasenius VM, Heikkil a P, Lundin J, Jackson DG, Joensuu H. High LYVE-1-positive lymphatic vessel numbers are associated with poor outcome in breast cancer. Clin Cancer Res 2004;10: Vleugel MM, Bos R, van der Groep P, Greijer AE, Shvarts A, Stel HV, van der Wall E, van Diest PJ. Lack of lymphangiogenesis during breast carcinogenesis. J Clin Pathol 2004;57: Nakamura Y, Yasuoka H, Tsujimoto M, Imabun S, Nakahara M, Nakao K, Nakamura M, Mori I, Kakudo K. Lymph vessel density correlates with nodal status. VEGF-C expression, and prognosis in breast cancer. Breast Cancer Res Treat 2005;91: Zhang GH, Yang WT, Zhou XY, Zeng Y, Lu HF, Shi DR. Study of the correlation between HER-2 gene and lymphangiogenesis and their prognostic significance in human breast cancer. Zhonghua Yi Xue Za Zhi 2007;87: Van der Schaft DW, Pauwels P, Hulsmans S, Zimmermann M, van de Poll-Franse LV, Griffioen AW. Absence of lymphangiogenesis in ductal breast cancer at the primary tumor site. Cancer Lett 2007; 254: Mohammed RA, Ellis IO, Elsheikh S, Paish EC, Martin SG. Lymphatic and angiogenic characteristics in breast cancer: morphometric analysis and prognostic implications. Breast Cancer Res Treat 2009; 113: Fernandez MI, Bolenz C, Trojan L, Steidler A, Weiss C, Alken P, Grobholz R, Michel MS. Prognostic implications of lymphangiogenesis in muscle-invasive transitional cell carcinoma of the bladder. Eur Urol 2008;53: Gombos Z, Xu X, Chu CS, Zhang PJ, Acs G. Peritumoral lymphatic vessel density and vascular endothelial growth factor C expression in early-stage squamous cell carcinoma of the uterine cervix. Clin Cancer Res 2005;11: Longatto-Filho A, Pinheiro C, Pereira SM, Etlinger D, Moreira MA, Jube LF, Queiroz GS, Baltazar F, Schmitt FC. Lymphatic vessel density and epithelial D2 40 immunoreactivity in pre-invasive and invasive lesions of the uterine cervix. Gynecol Oncol 2007;107: Mou JH, Yan XC, Li ZP, Wang D, Duan GJ, Xiang DB, Xiao HL, Zhang QH. Characteristic and clinicopathologic significance of lymphangiogenesis in colorectal cancer. Zhonghua Bing Li Xue Za Zhi 2005;34: Yang X, Li LR, Pan ZZ, Zhou ZW, Wan DS. Lymphatic vessel density in Dukes B rectal carcinoma and its correlation to prognosis. Ai Zheng 2006;25: Chen W, Shen W, Chen M, Cai G, Liu X. Study on the relationship between lymphatic vessel density and distal intramural spread of rectal cancer. Eur Surg Res 2007;39: Yan G, Zhou XY, Cai SJ, Zhang GH, Peng JJ, Du X. Lymphangiogenic and angiogenic microvessel density in human primary sporadic colorectal carcinoma. World J Gastroenterol 2008;14: Longatto-Filho A, Pinheiro C, Ferreira L, Scapulatempo C, Alves VA, Baltazar F, Schmitt F. Peritumoural, but not intratumoural, lymphatic vessel density and invasion correlate with colorectal carcinoma poor-outcome markers. Virchows Arch 2008;452: Gao J, Knutsen A, Arbman G, Carstensen J, Frånlund B, Sun XF. Clinical and biological significance of angiogenesis and lymphangiogenesis in colorectal cancer. Dig Liver Dis 2009;41: Stefansson IM, Salvesen HB, Akslen LA. Vascular proliferation is important for clinical progress of endometrial cancer. Cancer Res 2006;66: Kitadai Y, Kodama M, Cho S, Kuroda T, Ochiumi T, Kimura S, Tanaka S, Matsumura S, Yasui W, Chayama K. Quantitative analysis of lymphangiogenic markers for predicting metastasis of human gastric carcinoma to lymph nodes. Int J Cancer 2005;115: Nakamura Y, Yasuoka H, Tsujimoto M, Kurozumi K, Nakahara M, Nakao K, Kakudo K. Importance of lymph vessels in gastric cancer: a prognostic indicator in general and a predictor for lymph node metastasis in early stage cancer. J Clin Pathol 2006;59: Wang TB, Deng MH, Qiu WS, Dong WG. Association of serum vascular endothelial growth factor-c and lymphatic vessel density with lymph node metastasis and prognosis of patients with gastric cancer. World J Gastroenterol 2007;13: Yuanming L, Feng G, Lei T, Ying W. Quantitative analysis of lymphangiogenic markers in human gastroenteric tumor. Arch Med Res 2007;38: Gao P, Zhou GY, Zhang QH, Xiang L, Zhang SL, Li C, Sun YL. Clinicopathological significance of peritumoral lymphatic vessel density in gastric carcinoma. Cancer Lett 2008;263: Thelen A, Scholz A, Benckert C, Weichert W, Dietz E, Wiedenmann B, Neuhaus P, Jonas S. Tumor-associated lymphangiogenesis correlates with lymph node metastases and prognosis in hilar cholangiocarcinoma. Ann Surg Oncol 2008;15: Beasley NJ, Prevo R, Banerji S, Leek RD, Moore J, van Trappen P, Cox G, Harris AL, Jackson DG. Intratumoral lymphangiogenesis and lymph node metastasis in head and neck cancer. Cancer Res 2002;62: Maula SM, Luukkaa M, Grenman R, Jackson D, Jalkanen S, Ristam aki R. Intratumoral lymphatics are essential for the metastatic spread and prognosis in squamous cell carcinomas of the head and neck region. Cancer Res 2003;63: Franchi A, Gallo O, Massi D, Baroni G, Santucci M. Tumor lymphangiogenesis in head and neck squamous cell carcinoma: a morphometric study with clinical correlations. Cancer 2004;101: Kyzas PA, Geleff S, Batistatou A, Agnantis NJ, Stefanou D. Evidence for lymphangiogenesis and its prognostic implications in head and neck squamous cell carcinoma. J Pathol 2005;206: Straume O, Jackson DG, Akslen LA. Independent prognostic impact of lymphatic vessel density and presence of low-grade lymphangiogenesis in cutaneous melanoma. Clin Cancer Res 2003;9: Dadras SS, Paul T, Bertoncini J, Brown LF, Muzikansky A, Jackson DG, Ellwanger U, Garbe C, Mihm MC, Detmar M. Tumor lymphangiogenesis: a novel prognostic indicator for cutaneous melanoma metastasis and survival. Am J Pathol 2003;162: Giorgadze TA, Zhang PJ, Pasha T, Coogan PS, Acs G, Elder DE, Xu X. Lymphatic vessel density is significantly increased in melanoma. J Cutan Pathol 2004;31:672 7.

8 2754 SLEEMAN AND THIELE 64. Ohta Y, Shridhar V, Bright RK, Kalemkerian GP, Du W, Carbone M, Watanabe Y, Pass HI. VEGF and VEGF type C play an important role in angiogenesis and lymphangiogenesis in human malignant mesothelioma tumours. Br J Cancer 1999;81: Li Q, Dong X, Gu W, Qiu X, Wang E. Clinical significance of co-expression of VEGF-C and VEGFR-3 in non-small cell lung cancer. Chin Med J (Engl) 2003;116: Renyi-Vamos F, Tovari J, Fillinger J, Timar J, Paku S, Kenessey I, Ostoros G, Agocs L, Soltesz I, Dome B. Lymphangiogenesis correlates with lymph node metastasis, prognosis, and angiogenic phenotype in human non-small cell lung cancer. Clin Cancer Res 2005;11: Wang Y, Sun JG, Ye MF, Chen ZT. Clinical and prognostic significance of podoplanin-positive lymphatic vessel density in non-small cell lung carcinoma. Zhonghua Jie He He Hu Xi Za Zhi 2006;29: Faoro L, Hutto JY, Salgia R, El-Zayaty SA, Ferguson MK, Cheney RT, Reid ME, Armato SG, III, Krausz T, Husain AN. Lymphatic vessel density is not associated with lymph node metastasis in non-small cell lung carcinoma. Arch Pathol Lab Med 2008;132: Brundler MA, Harrison JA, de Saussure B, de Perrot M, Pepper MS. Lymphatic vessel density in the neoplastic progression of Barrett s oesophagus to adenocarcinoma. J Clin Pathol 2006;59: Mori D, Yamasaki F, Shibaki M, Tokunaga O. Lateral peritumoral lymphatic vessel invasion can predict lymph node metastasis in esophageal squamous cell carcinoma. Mod Pathol 2007;20: Inoue A, Moriya H, Katada N, Tanabe S, Kobayashi N, Watanabe M, Okayasu I, Ohbu M. Intratumoral lymphangiogenesis of esophageal squamous cell carcinoma and relationship with regulatory factors and prognosis. Pathol Int 2008;58: Saad RS, Lindner JL, Liu Y, Silverman JF. Lymphatic vessel density as prognostic marker in esophageal adenocarcinoma. Am J Clin Pathol 2009;131: Sedivy R, Beck-Mannagetta J, Haverkampf C, Battistutti W, H onigschnabl S. Expression of vascular endothelial growth factor-c correlates with the lymphatic microvessel density and the nodal status in oral squamous cell cancer. J Oral Pathol Med 2003;32: Mu~noz-Guerra MF, Marazuela EG, Martın-Villar E, Quintanilla M, Gamallo C. Prognostic significance of intratumoral lymphangiogenesis in squamous cell carcinoma of the oral cavity. Cancer 2004;100: Miyahara M, Tanuma J, Sugihara K, Semba I. Tumor lymphangiogenesis correlates with lymph node metastasis and clinicopathologic parameters in oral squamous cell carcinoma. Cancer 2007;110: Hinojar-Gutierrez A, Fernandez-Contreras ME, Gonzalez-Gonzalez R, Fernandez-Luque MJ, Hinojar-Arzadun A, Quintanilla M, Gamallo C. Intratumoral lymphatic vessels and VEGF-C expression are predictive factors of lymph node relapse in T1-T4 N0 laryngopharyngeal squamous cell carcinoma. Ann Surg Oncol 2007;14: Zhao D, Pan J, Li XQ, Wang XY, Tang C, Xuan M. Intratumoral lymphangiogenesis in oral squamous cell carcinoma and its clinicopathological significance. J Oral Pathol Med 2008;37: Birner P, Schindl M, Obermair A, Plank C, Breitenecker G, Kowalski H, Oberhuber G. Lymphatic microvessel density in epithelial ovarian cancer: its impact on prognosis. Anticancer Res 2000;20: Rubbia-Brandt L, Terris B, Giostra E, Dousset B, Morel P, Pepper MS. Lymphatic vessel density and vascular endothelial growth factor- C expression correlate with malignant behavior in human pancreatic endocrine tumors. Clin Cancer Res 2004;10: Sipos B, Kojima M, Tiemann K, Klapper W, Kruse ML, Kalthoff H, Schniewind B, Tepel J, Weich H, Kerjaschki D, Kl oppel G. Lymphatic spread of ductal pancreatic adenocarcinoma is independent of lymphangiogenesis. J Pathol 2005;207: Trojan L, Michel MS, Rensch F, Jackson DG, Alken P, Grobholz R. Lymph and blood vessel architecture in benign and malignant prostatic tissue: lack of lymphangiogenesis in prostate carcinoma assessed with novel lymphatic marker lymphatic vessel endothelial hyaluronan receptor (LYVE-1). J Urol 2004;172: Zeng Y, Opeskin K, Horvath LG, Sutherland RL, Williams ED. Lymphatic vessel density and lymph node metastasis in prostate cancer. Prostate 2005;65: Roma AA, Magi-Galluzzi C, Kral MA, Jin TT, Klein EA, Zhou M. Peritumoral lymphatic invasion is associated with regional lymph node metastases in prostate adenocarcinoma. Mod Pathol 2006; 19: Cheng L, Bishop E, Zhou H, Maclennan GT, Lopez-Beltran A, Zhang S, Badve S, Baldridge LA, Montironi R. Lymphatic vessel density in radical prostatectomy specimens. Hum Pathol 2008;39: De la Torre NG, Buley I, Wass JA, Turner HE. Angiogenesis and lymphangiogenesis in thyroid proliferative lesions: relationship to type and tumour behaviour. Endocr Relat Cancer 2006;13: Horiguchi A, Ito K, Sumitomo M, Kimura F, Asano T, Hayakawa M. Intratumoral lymphatics and lymphatic invasion are associated with tumor aggressiveness and poor prognosis in renal cell carcinoma. Urology 2008;71: Bolenz C, Fernandez MI, Trojan L, Hoffmann K, Herrmann E, Steidler A, Weiss C, Str obel P, Alken P, Michel MS. Lymphangiogenesis occurs in upper tract urothelial carcinoma and correlates with lymphatic tumour dissemination and poor prognosis. BJU Int 2009;103: Van den Eynden GG, Van der Auwera I, Van Laere SJ, Huygelen V, Colpaert CG, van Dam P, Dirix LY, Vermeulen PB, Van Marck EA. Induction of lymphangiogenesis in and around axillary lymph node metastases of patients with breast cancer. Br J Cancer 2006; 95: He Y, Rajantie I, Ilmonen M, Makinen T, Karkkainen MJ, Haiko P, Salven P, Alitalo K. Preexisting lymphatic endothelium but not endothelial progenitor cells are essential for tumor lymphangiogenesis and lymphatic metastasis. Cancer Res 2004;64: Religa P, Cao R, Bjorndahl M, Zhou Z, Zhu Z, Cao Y. Presence of bone marrow-derived circulating progenitor endothelial cells in the newly formed lymphatic vessels. Blood 2005;106: Schledzewski K, Falkowski M, Moldenhauer G, Metharom P, Kzhyshkowska J, Ganss R, Demory A, Falkowska-Hansen B, Kurzen H, Ugurel S, Geginat G, Arnold B, et al. Lymphatic endothelium-specific hyaluronan receptor LYVE-1 is expressed by stabilin-11. F4/ 801, CD11b1 macrophages in malignant tumours and wound healing tissue in vivo and in bone marrow cultures in vitro: implications for the assessment of lymphangiogenesis. J Pathol 2006;209: Jiang S, Bailey AS, Goldman DC, Swain JR, Wong MH, Streeter PR, Fleming WH. Hematopoietic stem cells contribute to lymphatic endothelium. PLoS ONE 2008;3:e Maruyama K, Ii M, Cursiefen C, Jackson DG, Keino H, Tomita M, Van Rooijen N, Takenaka H, Damore PA, Stein-Streilein J, Losordo DW, Streilein JW. Inflammation-induced lymphangiogenesis in the cornea arises from CD11b-positive macrophages. J Clin Invest 2005; 115: Kerjaschki D, Huttary N, Raab I, Regele H, Bojarski-Nagy K, Bartel G, Krober SM, Greinix H, Rosenmaier A, Karlhofer F, Wick N, Mazal PR. Lymphatic endothelial progenitor cells contribute to de novo lymphangiogenesis in human renal transplants. Nat Med 2006;12: Krishnan J, Kirkin V, Steffen A, Hegen M, Weih D, Tomarev S, Wilting J, Sleeman JP. Differential in vivo and in vitro expression of vascular endothelial growth factor (VEGF)-C and VEGF-D in tumors and its relationship to lymphatic metastasis in immunocompetent rats. Cancer Res 2003;63: Cao R, Bjorndahl MA, Religa P, Clasper S, Garvin S, Galter D, Meister B, Ikomi F, Tritsaris K, Dissing S, Ohhashi T, Jackson DG, et al. PDGF-BB induces intratumoral lymphangiogenesis and promotes lymphatic metastasis. Cancer Cell 2004;6: Iwata C, Kano MR, Komuro A, Oka M, Kiyono K, Johansson E, Morishita Y, Yashiro M, Hirakawa K, Kaminishi M, Miyazono K. Inhibition of cyclooxygenase-2 suppresses lymph node metastasis via reduction of lymphangiogenesis. Cancer Res 2007;67: Shibata MA, Morimoto J, Shibata E, Otsuki Y. Combination therapy with short interfering RNA vectors against VEGF-C and VEGF-A suppresses lymph node and lung metastasis in a mouse immunocompetent mammary cancer model. Cancer Gene Ther 2008;15: Da MX, Wu Z, Tian HW. Tumor lymphangiogenesis and lymphangiogenic growth factors. Arch Med Res 2008;39: Thiele W, Sleeman JP. Tumor-induced lymphangiogenesis: a target for cancer therapy? J Biotechnol 2006;124: Eccles S, Paon L, Sleeman J. Lymphatic metastasis in breast cancer: importance and new insights into cellular and molecular mechanisms. Clin Exp Metastasis 2007;24: Mandriota SJ, Jussila L, Jeltsch M, Compagni A, Baetens D, Prevo R, Banerji S, Huarte J, Montesano R, Jackson DG, Orci L, Alitalo K, et al. Vascular endothelial growth factor-c-mediated lymphangiogenesis promotes tumour metastasis. EMBO J 2001;20: Skobe M, Hawighorst T, Jackson DG, Prevo R, Janes L, Velasco P, Riccardi L, Alitalo K, Claffey K, Detmar M. Induction of tumor lymphangiogenesis by VEGF-C promotes breast cancer metastasis. Nat Med 2001;7: Stacker SA, Caesar C, Baldwin ME, Thornton GE, Williams RA, Prevo R, Jackson DG, Nishikawa S, Kubo H, Achen MG. VEGF-D promotes the metastatic spread of tumor cells via the lymphatics. Nat Med 2001;7: He Y, Kozaki K, Karpanen T, Koshikawa K, Yla-Herttuala S, Takahashi T, Alitalo K. Suppression of tumor lymphangiogenesis and lymph node metastasis by blocking vascular endothelial growth factor receptor 3 signaling. J Natl Cancer Inst 2002;94: Kopfstein L, Veikkola T, Djonov VG, Baeriswyl V, Schomber T, Strittmatter K, Stacker SA, Achen MG, Alitalo K, Christofori G. Distinct roles of vascular endothelial growth factor-d in lymphangiogenesis and metastasis. Am J Pathol 2007;170: