The role of lymphangiogenesis and angiogenesis in tumor metastasis

The main function of blood vessels is to transport oxygenated blood, to exchange oxygen, carbon dioxide, water and mineral salts between blood and tissues and to regulate the pressure of the flow in the closed system powered by the heart. The lymphatic system, on the other hand, begins in peripheral tissues with blind-ended capillaries and has an open, semicircular layout. Lymph flows unidirectionally from the peripheral tissues to the blood. It does not carry oxygen or essential nutrients. Its primary function is to absorb extravasated protein-rich fluids, lipids, macromolecules and immunocompetent cells from the interstitial spaces within tissues. After resorption from the initial ducts (lymphatic capillaries and then pre-collectors), lymph is transported to larger vessels (lymphatic collectors and trunks) and flows back into the bloodstream mainly via the left lymphatic duct (thoracic duct). Normal functioning lymphatic vessels thus maintain plasma volume, prevent increases in tissue pressure and allow easy passage of leukocytes, thereby playing an important role in the proper functioning of the immune system and the immune surveillance of the whole body [4, 7‐9].

Although blood and lymphatic vessels share a common embryological origin, they differ significantly. A first essential difference is the anatomy of lymphatic and blood vessels. Initial and terminal lymphatic capillaries are 10–60 μm in diameter and are lined with a layer of lymphatic endothelial cells (LECs) [10]. Blood capillaries are approximately 5–20 μm in diameter and have a uniform, compact layer of endothelium. Lymphatic capillaries, unlike blood capillaries, have an incomplete discontinuous basal lamina, or no basal lamina at all, and lack pericytes and smooth muscle cells [4, 8, 10, 11]. An important feature of lymphatic capillaries is the size of their lumen, which is three-fold wider than that of blood capillaries. Moreover, lymphatic capillaries are unique in that they have reticular, elastic and collagen fibers (anchoring filaments), which bind LECs to the extracellular matrix (ECM), a property that is vital to a proper lymph flow. The fibers stretch to open the lymphatic lumen (intracellular space) when the volume of the interstitial fluid increases, thus producing hydrostatic pressure. After this, the interstitial fluid flows into the lymphatic system causing the capillaries to dilate rather than to collapse [4, 7, 10‐12]. This is a major feature of lymph absorption. The lymphatic collectors and trunks differ, however, structurally form lymphatic capillaries and are histologically similar to veins. They have a thin, three-layered coat and valves at the jugulosubclavian junction, which prevents blood reflux to lymphatic ducts as also retrograde lymph flow [8, 10, 13]. Normal blood vessels also contain three layers. The innermost layer (tunica intima) consists of a single layer of endothelial cells surrounded by connective tissue called the internal elastic lamina. The middle layer (tunica media) is built up of a basement membrane and smooth muscle cells surrounded by the external elastic lamina. The outermost layer (tunica adventitia) consist of connective tissue containing nerves that innervate the vessel [14]. Pathological veins that are formed as a result of tumor neovascularization differ significantly from normal veins. They are characterized by a chaotic structure in which endothelial cells do not adhere tightly to each other but, instead, form protrusions towards the lumen of the vessel. Also the pericytes adhere only loosely to the endothelium. The basal lamina is thinner than in normal vessels and has many fenestrations, which makes the vessels permeable. The diameter of the vessels may vary and blood clots can be formed, which may result in local differences in blood pressure. Increases in blood pressure and a high permeability of the vessels may result in the appearance of exudates, which prohibit the intravasation of cells. A chaotic organization of the vessel networks with numerous blind-ended vessels may lead to blood stasis, or even backflow [15, 16].

Taking the above considerations into account, it appears that the lymphatic vessel pathway provides a better and safer route for cancer cell dissemination than the blood vessel pathway. The composition of the lymph fluid is almost identical to that of the interstitial tissue fluids, which promotes the survival of migrating tumor cells. Moreover, the discontinuous structure of the lymph capillary elements, a low lymph flow, a minimalized shear stress and a high concentration of hyaluronic acid, which plays an important role in cell protection and survival, give the lymphatic system an advantage over the bloodstream, in which mechanical forces and the toxicity of pure serum may negatively affect circulating tumor cells [7, 17]. Nevertheless, before an unequivocal claim can be made as to which route of dissemination is more effective, two questions need to be answered. The first one deals with the energy that metastasizing cells require for migration, i.e., whether it is more energy-efficient for cells to actively move to secondary sites or be shed passively. The second question is whether it is more proficient, from the cell’s point of view, to choose the hematogenous or the lymphatic pathway for the effective formation of distant micro-metastases. Lymphatic vessels are relatively leaky compared to blood vessels and, as such, are considered essential for tumor cell spread. The structure of blood vessels would, under normal circumstances, force tumor cells to spend more energy during intra- and especially extravasation. In the tumor neovasculature, however, many abnormalities can be found that actually facilitate tumor cell migration, including disorganized wall structures, endothelial fenestrations and a thin or even non-existing basement membrane [18]. The fragile new blood vessels that vascularize primary tumors are, to a certain extent, comparable to leaky lymphatic capillaries that lack a continuous basement membrane and contain many open junctions and pores. This open blood vessel structure suggests that tumor intravasation may be a passive process. However, it is well documented that CD73, an enzyme converting AMP to adenosine, is actively involved in vascular permeabilization. And although the ATP metabolism differs in blood and lymphatic vessels, it has been found that CD73 also plays an important role in normal lymphocyte migration into lymph nodes [19]. This enzyme may be considered as a major factor in the metastasis of tumor cells. Initially, tumor cells grow in their primary niche, but when the tumor reaches a diameter of about 1 mm, it invades its microenvironment and collapses the newly formed tumor blood vessels. Their fragile non-linear/disordered structure gives way under the pressure of the tumor mass, enabling passive entry of tumor cells into the vessel’s lumen and, subsequently, metastasis [20]. On the other hand, it has been shown that invasive tumor cells may detach form their primary masses and enter tumor-associated absorbing lymphatic (TAAL) vessels through intra-endothelial channels (1.8–2.1 μm in diameter), thus taking a passive route into the lymphatic circulation [21]. To simplify this model, tumor cells may be washed out from the tumor mass and, with the tide of tissue fluid, be pushed into lymphatic drainage canals, thereby initiating invasion. The passive model of intravasation is supported by the finding that most shed cells are non-vital and non-clonogenic. This means that the only cells that can survive in vessels (i.e., withstand the mechanical stresses and the attack of immune cells) and form metastases are tumor stem cells and cells that express a metastatic phenotype. However, such an argument implies the acceptance of an active mechanism of metastasis [21‐23]. Other arguments in favor of an involvement of active mechanisms in the initial steps of metastasis include the accumulation of mutations, changes in the expression of adhesion molecules, the presence of chemokine gradients, expression of the urokinase plasminogen activator (uPA) followed by activation of metalloproteinases (MMPs), and the supportive role of stromal cells and cancer-associated fibroblasts, CAFs [24]. It is believed that alterations in the primary tumor microenvironment/niche induce tumor cells to migrate towards blood or lymphatic vessels. Active migration and entry into the vessels may also be facilitated by changes in the organization of the cytoskeleton as well as the acquisition of a metastatic phenotype through epithelial-mesenchymal transition (EMT) [23].

Despite many reports in the literature on this subject, there is currently no unequivocal answer to the question which conditions determine the choice of an active or a passive route of migration by tumor cells. It seems that, depending on the type of tumor, the stage of its development and/or the metastatic target organ, tumor cells may select either one or both of these routes.

Besides the acquisition of specific genomic changes by tumor cells that enable their growth and survival, epithelial-mesenchymal transition (EMT) and the reverse mesenchymal-epithelial transition (MET) are generally considered as some of the most fundamental processes underlying cancer cell dissemination. EMT is usually reported as being essential for the initial stages of malignant transformation and tumor development at the primary site, whereas MET is believed to be pivotal for the later, metastatic stages and for the formation of secondary tumors at distant sites [25]. Tumor cells undergo “type III EMT”, which differs from “type II EMT” that occurs during inflammation or fibrosis and “type I EMT”, which takes place under normal physiological conditions such as wound healing [26‐28]. During EMT tumor cells acquire a mesenchymal, migratory and invasive phenotype by losing their intercellular junctions (i.e., adherens junctions, tight junctions, desmosomal junctions and also, partially, gap junctions), typical molecular markers (E-cadherin, cytokeratins), cytoskeletal organization (changes in microtubules, actin filaments, β-filamin or talin), apical/basolateral polarity and, finally, contact inhibition [27, 29‐32]. At their primary site, tumor cells receive EMT promoting signals from activated stroma, while at metastatic sites such signals are weak or absent. Under the latter circumstances, metastatic tumor cells convert to an epithelial phenotype via MET. This process is also believed to support tumor-normal cell interactions at distant, metastatic sites, and to be facilitated by the stimulatory activity of the target organ parenchyma that induces the re-expression of E-cadherin. As a consequence, connections can be formed between neoplastic and normal cells. Such connections are very important for tumor cell survival within the new microenvironment [32]. On the basis of direct and indirect, paracrine stimulations, tumor cells may enter a state of dormancy at the metastatic target site. Dormant cells are characterized by a low metabolic activity, a suppressed anoikis associated with the formation of cell heterotypic E-cadherin, and a resistance to cytostatics due to activation of the receptor tyrosine kinase ErbB4 and induction of the PI3K-Akt pathway [25, 33‐35]. Obviously, this is a simplified model of EMT-MET, and there are other factors that significantly promote not only EMT-MET, but also lymphangiogenesis and angiogenesis. Among them, the most important ones are α-SMA, whose increased expression by myofibroblasts has been associated with a high expression of N-cadherin, a LYVE-1-positive vessel count, and increased expression of VEGF, stromal cell-derived factor 1 (SDF-1) also known as C-X-C motif chemokine 12 (CXCL12), insulin-like growth factor-2 (IGF-2), hypoxia-inducible factor-1α (HIF-1α), transforming growth factor β (TGF-β) and hepatocyte growth factor (HGF) [30, 36‐39]. In addition, immune cells such as macrophages, myeloid-derived suppressor cells (MDSCs), mast cells and neutrophils may contribute to EMT/MET transitions and the formation of new vessels through the production of cytokines, growth factors or proteases [30]. On the other hand, there are also molecules that may inhibit tumor angiogenesis, lymphangiogenesis and invasion. One such molecule is KAI-1/CD82, which belongs to the tetraspanin family of proteins and is considered to be a metastasis suppressor. This molecule is localized on the cell membrane and interacts with integrins and chemokines responsible for the adhesion, signaling and mobility of cells. Decreased levels of KAI-1/CD82 have been linked to limited cancer cell invasiveness and the suppression of metastatic cell growth mainly through inhibition of β-catenin-mediated EMT [40, 41]. Generally, molecules that are involved in EMT/MET transitions can be classified as EMT inducers (upstream cytokines and growth factors or receptors that initiate transition), EMT regulators (downstream transcription factors controlling the transition process) and EMT effectors (molecules that cause cell phenotype changes and endow cells with an invasive character) [32, 42].

Although EMT and MET are widely recognized as being essential for tumor cell metastasis, they may proceed differently in different cancers due to diverse characteristics of the respective blood and lymphatic vessel systems. It has e.g. been shown that prostate and breast cancers isolated form sentinel lymph nodes exhibit an increased invasive potential without any upregulation of mesenchymal markers [43, 44]. This finding confirms the concept that although mesenchymal transition contributes to the successful metastasis of tumor cells, lymph node dissemination does not per se require EMT [45]. This notion may be explained by the fact that the structure of the lymphatic system does not force mesenchymal cells to increase their invasive phenotype. Moreover, EMT facilitates intravasation, whereas cells leaving the vessels (extravasation) do not require this process [46]. Therefore, it may be concluded that in contrast to blood vessel dissemination, successful lymphatic migration may not require EMT. As such, EMT does not always appropriately imply the invasion of tumor cells and, although crucial, it is only one of the possible mechanisms underlying tumor cell metastasis.

The tumor stroma is composed of non-cellular components of the extracellular matrix (ECM) such as proteins, glycoproteins, proteoglycans and polysaccharides endowing complex physical and biological properties to the stroma, as well as immune cells, endothelial cells and fibroblasts (CAFs) that participate in the early stages of tumor cell dissemination [47, 48]. The ECM, besides its structural and biomechanical features, can also function as a repository for active components, such as growth factors, which are stored and released during alteration or remodeling of its composition. Some of the most important constituents of the ECM are laminins, which have a significant impact on cellular dynamics, and collagens, which are the major structural components of the matrix [49‐51]. Reorganization of these and other matrix constituents during cancer development results in deregulation of the ECM and disruption of its integrity and architecture, thereby promoting epithelial cell transformation and tumor cell progression [48]. Deregulated ECM dynamics are closely related to the expression and activity of ECM enzymes including MMPs, heparanases, 6-O-sulfatases, cysteine cathepsins, urokinase and the serine protease plasmin [52, 53]. This deregulation leads to essential changes in ECM properties, which not only may induce tumor cell motility, but may also affect tumor micro-environmental stromal cells such as CAFs, immune cells and mesenchymal stem cells (MSCs) [54‐57].

It has been found that released or plasma fibronectins play an important role in tumor cell adhesion, migration, invasion and survival by activating integrins via the MAPK/ERK pathway [58]. Other ECM components and receptors, including heparan sulfate proteoglycans and CD44, may facilitate the growth and motility of tumor cells [59‐61]. An abnormal ECM has also been found to participate in the induction of angiogenesis and lymphangiogenesis during tumor progression. It has been reported that fragments of type IV and type XVIII ECM collagens, including endostatin, tumstatin, canstatin, arresten and hexastatin, strongly influence angiogenesis, either directly or indirectly by modulating the VEGF level [62]. They may also modify lumen entrapment involved in tube formation during angiogenesis. [63, 64]. The role of the ECM in lymph vessel formation is as yet poorly recognized, but it has been shown that the ECM receptor integrin α9β1 may be involved in the induction of tumor lymphangiogenesis [65, 66]. Similarly, low molecular weight hyaluronian (LMW-HA) has been found to promote lymphangiogenesis via interactions with its lymphatic vessel endothelial hyaluronian receptor 1 (LYVE-1), which leads to the induction of LEC proliferation and tube formation [67]. Recently, it has also been found that MT1-MMP-mediated proMMP-2 activation and the expression of ECM1 soluble protein and EMILIN1, an elastic microfibryl-associated protein, are closely linked to lymphangiogenesis and tumor invasion [68‐70]. Taken together, it may be concluded that changes in biochemical and biomechanical properties of the ECM represent important factors affecting tumor cell behavior during metastasis.

If tumor cells can use passive and active mechanisms of intravasation, the question arises what the basis is of dissemination via blood and lymphatic vessels. It has already been reported that carcinomas and melanomas are more likely to form lymph node metastases than sarcomas [46]. It is unclear, however, why, when or where the decision about the route of intravasation is made. Some hypotheses on this matter have already been put forward and all of them appear to be equally probable. The most obvious factors that may be involved in this process are the physical/mechanical conditions and the genetic or epigenetic programs that are innate to tumor cells. Also, the role of factors that attract tumor cells towards blood or lymphatic vessels should not be ignored. Among them are inflammatory and host hematopoietic precursors, but also soluble factors such as chemokines, growth factors and soluble receptors [71]. They not only affect the metastatic phenotype of tumor cells, but also in cases where due to various reasons (e.g. receptor mismatch, difficulties with EMT, a migration mode that makes it difficult to penetrate the blood wall barrier) tumor cells are unable to cross blood vessels, they direct them into the peri-tumoral lymphatic system (metachronous seeding), which provides a safer and easier way for the cells to intravasate and disseminate. As an example, it has been reported that bradykinin may act as a signal that attracts glioma cells to blood vessels [72]. Subsequently, these tumor cells may establish so-called satellite lymph node metastases, which disseminate metastatic cells via the thoracic duct. Some authors claim that lymph nodes select tumor cells, enabling those with a high enough malignant phenotype to disseminate further. They explain ipso facto the differences in malignancy between primary tumor cells and metastatic tumor cells [46]. Following this idea, it seems obvious that inhibition of lymph node metastasis should inhibit hematogenous spread. Experimental data show, however, that this is not always the case. Moreover, it has been reported that distant metastases can be formed despite a lack of metastatic cells in sentinel and distant lymph nodes. This, in turn, may confirm direct dispersal of tumor cells into blood vessels. There is also a model which proposes that tumor cells may stay for some time in a non-metastatic state. This state lasts until the cells are activated and recruited to disseminate simultaneously via blood and lymphatic vessels [46]. This hypothesis may explain the quick and massive metastasis which is characteristic for some cancers.

Tumor cells may disseminate via blood or lymphatic vessels, but do they show a “predilection” for one route of migration over the other? Such predilection may depend on various factors that are specific for the tumor cells, as well as for their microenvironment and the newly formed vessels. In addition, specific molecular signaling pathways may play a major role. Differences in gene expression between the lymphatic and blood endothelium may constitute one of the major factors that is decisive for the route of dissemination that tumor cells choose. Blood endothelial cells (BECs) typically express CD44, ICAM1, Tie-2/Ang-1 VEGFR-1 and -2, Neutropilin-1 receptors for VEGF-A, -C and -D, and secrete IL-6/8 and MCP-1. On the other hand, lymphatic endothelial cells express c-Met/HGF, Tie-2/Ang-1/2, IGF-Rs/IGF-1/2, FGF-Rs/FGF-2, Podoplanin, LYVE-1 and VEGFR-2 and -3, receptors for VEGF-C and -D [73‐75]. The role of these factors is widely accepted now, despite controversies on the role of VEGF-D in lymphangiogenesis and tumor cell dissemination via lymphatic vessels in some cancers, such as ovarian and breast cancers [76‐79]. VEGF-D has been reported to act as a factor that induces both intra- and peri-tumoral lymphatic vessel development, but not necessarily lymph node metastasis [80, 81].

Gene expression profiles may not only differentiate the properties of the two cell types involved (i.e., BECs and LECs), but also the physiological functions of blood and lymphatic vessels and their potential to be selected by tumor cells as a route for metastasis [74]. On the other hand, selection pressure can also be exerted on tumor cells through the expression of different receptors and signaling molecules by the lymphatic or blood endothelium, which allows cells to transmigrate via the blood or lymphatic vessel linings only, depending on what specific co-receptors the tumor cells express. It has also been suggested that the choice between lymphangiogenesis and angiogenesis may depend on the ratio of the different inducing factors present within the local tumor microenvironment [82]. Also, crosstalk between lymphatic and blood endothelial cells, as well as between endothelial cells and the vessel milieu, should not be ignored as important aspects in the selection of one of the two routes of tumor cell dissemination [80]. It appears that the ultimate selection depends on several factors, including the specific structure and mechanical functionality of the vessels as also the expression of adhesion molecules, the secretion of chemokines and the activity of specific signaling pathways. Which pathway is chosen depends on the concentration of local factors at the primary site as also at the site of the metastatic niche, the tumor cell of origin, the stage of tumor development and, conceivably, the patient’s health status. It seems most probable that both routes may be involved in metastasis, but not necessarily at the same time (Fig. 1).

For a long time scientists were convinced that only blood vessels, which drain the whole body, are able to transport cancer cells to secondary, metastatic sites. This view changed when direct and indirect paracrine tumor-stroma interactions in the primary tumor niche, as well as intra-nodal lymphangiogenesis, were discovered [83]. It was found that tumor cells can alter the surrounding microenvironment and, thus, influence tumorigenesis through crosstalk with dendritic cells, CAFs, macrophages, lymphocytes and pericytes, all of which can secrete soluble molecules that exhibit angiogenic or lymphangiogenic activities [84]. These molecules may, in turn, stimulate the enlargement of tumor lymphatic vessels, thereby facilitating cancer cell invasion. Another milestone was the introduction of the lymphvascular niche concept [85]. This concept may explain the role of lymphatic vessel formation within nodes, thereby constituting an intermediate platform for the lymphatic metastasis of cancer cells. Indeed, it has experimentally been shown that primary tumors can induce lymphatic vessel formation (neo-lymphangiogenesis) within the tumor draining lymph node [86]. This process precedes metastasis, possibly indicating that primary tumors may first generate a favorable microenvironment at this site for a subsequent preferential and successful dissemination. This lymphvascular niche may not only attract spreading tumor cells but also a subset of cancer stem cells (CSCs), which are the potential initiators of secondary tumor development at distant sites [87‐89]. This notion is also of relevance for clinicians who use resected primary tumor samples and regional lymph nodes to determine the stage of the disease, the most optimal treatment regimen and the patient’s prognosis.

Another important point is the origin of the endothelial cells that form tumor lymphatic vessels. As yet, three potential sources of lymphatic endothelial cells (LECs, Fig. 2) have been reported [87]. The first one is a pre-existing lymphatic vessel in which, after appropriate stimulation, LECs proliferate and migrate leading to neo-lymphatic outgrowth. Subsequently, the tumor cells and the tumor stroma induce the formation of new lymphatic capillaries by secreting VEGF-C and other cytokines and chemokines. Subsequent interactions with specific receptors induce LEC-based tube formation by stimulating cell proliferation and longitudinal growth. Thus, tumor LECs may be induced locally, i.e., be derived from local vessels in specialized parts of the lymphatic system [87, 90]. A second route for the formation of tumor LECs is the transdifferentiation of endothelial cells from pre-existing blood vessels. In this process VEGF-C plays an essential role, as also the lymphatic-specific receptor VEGFR-3, which is expressed in blood vessels in tumors, and the key transcription factors SOX18, COUP-TFII and PROX-1 [87, 90, 91]. Likewise, it has been shown that integration of circulating cells that exhibit lymphendothelial features may initiate a pathologic outgrowth of the lymphatic system. Thus, a third source of tumor LECs comes from transdifferentiation of non-endothelial cells [92]. Although bone marrow-derived cells (BMDCs), including endothelial progenitor cells, are essential for the formation of new blood vessels, it is conceivable that they may also contribute to neo-lymphangiogenesis in tumors. Their participation may not only be direct via transdifferentiation, but they may also play inducing or supporting roles under pathological conditions, without necessarily taking part in the lymphangiogenic process itself [93]. Other cells, such as mesenchymal stem cells (MSCs) or tumor associated macrophages (TAMs), may also participate in this process. Under specific conditions prevailing in tumor masses such as hypoxia, MSCs may differentiate into ECs, thereby contributing not only to angiogenesis but also to lymphangiogenesis [94]. TAMs, which belong to the myeloid lineage, are multifunctional cells that, depending on the micro-environmental conditions, may switch their phenotype and functionality. They can transdifferentiate and structurally act as ECs, i.e., form cellular elements of the lymphatic vessel wall [95]. On the other hand, macrophages also constitute a rich source of bioactive molecules that, under appropriate conditions such as tumor inflammation, can be released by the activated cells. Among these molecules are VEGF-A/-C/-D, which initiate lymphangiogenesis both through the stimulation of LEC proliferation and the consecutive recruitment of TAMs. In this latter scenario, it is highly probable that both subtypes of macrophages (i.e., M1 and M2) may interact with tumor LECs and participate in pathologic lymphangiogenesis, either directly or indirectly in a paracrine fashion [87, 96‐100].

In blood vessels, a similar role is played by blood endothelial cells (BECs). Both BECs and LECs are endothelial cells, so it may be reasonable to assume that these two types of cells do not differ significantly. In conformity with the diverse roles played by these cells in blood and lymphatic vessels, however, they exhibit clear differences in cell-cell and cell-matrix interactions. In fact, differences have been found not only in their arrangements in the respective vessels, but also in their responses to the micro-environmental signals that they receive and secrete (i.e., angiocrine and lymphangiocrine factors). These functional differences may obviously result from the interstitial flow conditions to which the ECs are exposed, but it has also recently been shown that an important role is played by the genes they express [101‐104]. This is a third level at which the morphological features and the micro-environmental interactions of ECs may be programmed and controlled. Despite these differences, both BECs and LECs play important roles in blood and lymph flow and, under pathologic conditions, they may serve as enhancers of tumor cell adhesion to the vessel wall, the transmigration of tumor cells and, consequently, tumor progression.

Another intriguing question is to what extent intra-tumor lymphatic vessels (ITLs) and peri-tumor lymphatic vessels (PTLs) participate in tumor cell dissemination. Two types of tumor lymphatic vessels can be distinguished on basis of their localization in the cancerous mass. Lymphatic vessels, both pre-existing and newly formed, can be found in the tumor periphery and inside the tumor mass and are, accordingly, called peri- and intra-tumoral lymphatic vessels, respectively [105]. Although their roles in tumor dissemination are different, they are both considered to be responsible for the formation of metastases. ITLs have been associated with a poor survival, whereas PTLs have been associated with the occurrence of nodal metastases and overall clinical outcome [106‐108]. In primary colorectal tumors, immunohistochemical staining has revealed the presence of an extensive PTL network, which may be related to a relatively short time from tumor development to the formation of metastases [107]. PTLs are generally believed to uptake tumor cells and to facilitate their dissemination. ITLs are, on the other hand, usually small, compressed, collapsed and non-functional, due to increased mechanical forces related to tumor growth, the invasion of migrating tumor cells and an increased interstitial fluid pressure inside the tumor mass [87]. Therefore, ITLs may be considered as components within tumors that initiate and promote metastasis, but do not act as routes of direct cancer cell spread or enhanced lymph node metastasis (LNM). This role is attributed to PTLs, which surround the tumor periphery with functional vessels. By increasing the absorptive area, which collects fluid and tumor cells from the external layers of the cancer mass that mainly contain fast proliferating tumor cells, they promote lymphatic metastasis [109]. Such lymphatic vessels are sufficient for a cancer to spread, which is supported by the fact that tumors that lack ITLs are often still able to disseminate. Therefore, an increased PTL vessel density, especially in VEGF-C over-expressing tumors, is usually considered as a predictor of a high risk of lymphatic metastasis [110]. Additionally, when tumor cells penetrate PTLs they may, through direct interaction, stimulate the proliferation of normal LECs (NLECs) and, in this way, promote or enhance lymphangiogenesis. Obviously, paracrine influences of NLEC-derived lymphangiogenic factors are also important. Together, these factors may be potent enough to re-program NLECs to tumor LECs (TLECs), which exhibit different morphological, functional and molecular characteristics [1].

Many additional factors are known to be strongly related to tumor cell dissemination and, consequently, a poor prognosis, including lymphatic vessel density (LVD), blood vessel density (BVD), interstitial fluid pressure (IFP) and tumor hypoxia. Although these factors are not unique to tumor dissemination and/or a poor prognosis, they may occasionally constitute a therapeutic problem.

Dissemination of cancer cells to regional lymph nodes is the first step in metastasis and, as such, serves as a useful tool for cancer staging and prognosis [131, 132]. In general, lymph node metastasis correlates with a poor prognosis. Other, auxiliary factors that predict a poor outcome include micro-lymphatic vessel density (MLD) and high expression levels of VEGF-C, CXCR4, Flt-4, VEGFR-3 and VEGF-D, which have been proven to contribute to lymphatic involvement and nodal metastasis [133‐136]. Factors that regulate or alter the rate of lymphatic metastasis can be classified as endocrine, cytotoxic, anti-angiogenic, anti-inflammatory and immune modulatory. At a higher level, these factors can be divided into exogenous and endogenous, influencing stimulators or inhibitors of lymph node dissemination [103]. Whether lymph node metastases indeed fully correlate with a poor prognosis and whether they may constitute a prognostic value in predicting distant dissemination of tumor cells to other organs is still a matter of debate. On the one hand, it has been stated that lymph node metastasis may not be related to tumor aggressiveness and tumor cell migration to distant organs. This claim is based on two observations. Firstly, it has been found that lymphadenectomy may not provide survival benefits and that tumor cells from primary masses may exhibit a similar disseminating potential as those from lymph nodes. Secondly, it has been found that tumor cells that have entered and adapted to the lymphatic system may not be able to efficiently form organ-specific metastases [137]. An opposing view is that disseminating tumor cells may efficiently use lymph node blood vessels or efferent lymphatic vessels to spread to other parts of the body. In addition, dissemination to lymph nodes may also occur in the absence of typical lymphangiogenesis, since cancer cells may also employ pre-existing lymphatic vessels. Therefore, while the presence or absence of cancer-related lymphangiogenesis may depend on tumor type, migration into lymph nodes seems to be an indispensable element of effective metastasis [138]. The latter theories appear to closely reflect the actual situation, since a lack of nodes often means that no distant metastases can be formed, which indicates that transition of tumor cells via nodes is a prerequisite for further dissemination. Lymphangiogenesis and migration of tumor cells into lymph nodes seems to be a preferential, active process that is necessary for further dissemination. Tumor cell migration may also induce lymph node lymphangiogenesis (LNL). The concept of LNL suggests that lymphatic and distant metastases are closely connected with each other as well as with tumor-inherent behavior and local responses of the host immune system to tumor-derived stimuli [139].

The primo vascular system (PVS) is a recently discovered novel circulatory system that may exist next to the lymphatic and blood vascular systems. Initially, vasculogenic mimicry was considered to represent an additional relevant vascular structure, but its primitive micro-circulation failed to provide an explanation for potential routes of tumor cell metastasis. Therefore, additional studies were performed that finally led to the discovery of a third vascular compartment currently known as the primo vascular system [140‐142]. This system was found to be present throughout the whole body on organ surfaces, inside lymphatic and blood vessels and their sub-vessels, as well as on the surface, around and within subcutaneous tumors [140, 141, 143, 144]. This structure is anatomically composed of small primo vessels (PV) with diameters of 20–50 μm and primo nodes (PN) approximately 100–1000 μm in size [141, 143]. Because of its wide distribution, its high density in tumor masses and its connection with the tumor microenvironment, the PVS is currently considered as a potent route for cancer cell metastasis. Its role may be important especially since the PVS directly connects primary and secondary tumors and since cancer cells can actively be transported via this system [143]. Moreover, because the PVS has its own circulating fluid, which contains cells expressing stem cell markers (i.e., CD133, Oct4 or Nanog), it may play a role in the regeneration of cancer stem cells or serve as a unique niche for these cells [140, 143]. It may, therefore, be hypothesized that next to lymphatic and blood vessels, cancer cells use the PVS for effective dissemination and the formation of secondary tumors at distant sites. This hypothesis may at least partially explain the failure or ineffectiveness of previous and current clinical trials designed to inhibit metastasis by only suppressing lymphangiogenesis or angiogenesis [144]. It would also disprove the dogma of the existence of only two routes for cancer cell dissemination.