Histological and biochemical changes in lymphatic vessels after skeletal muscle injury induced by lengthening contraction in male mice.

Lymphatic vessels are actively involved in the recovery process of inflamed tissues. However, the changes in intramuscular lymphatic vessels during inflammation caused by skeletal muscle injury remain unclear. Therefore, the purpose of this study was to clarify the changes in lymphatic vessels after skeletal muscle injury. The left tibialis anterior muscles of male mice were subjected to lengthening contractions (LC) for inducing skeletal muscle injury, and samples were collected on Days 2, 4, and 7 for examining changes in both the skeletal muscles and intramuscular lymphatic vessels. With hematoxylin-eosin staining, the inflammatory response was observed in myofibers on Days 2 and 4 after LC, whereas regeneration of myofibers was found on Day 7 after LC. The number and area of intramuscular lymphatic vessels analyzed by immunohistochemical staining with an antibody against lymphatic vessel endothelial hyaluronan receptor 1 were significantly increased only on Day 4 after LC. Based on the abovementioned results, intramuscular lymphatic vessels undergo morphological changes such as increase under the state of muscle inflammation. This study demonstrated that the morphology of intramuscular lymphatic vessels undergoes significant changes during the initial recovery phase following skeletal muscle injury.

The role of lymphangiogenesis in cardiovascular diseases and heart transplantation.

Cardiac lymphangiogenesis plays an important physiological role in the regulation of interstitial fluid homeostasis, inflammatory, and immune responses. Impaired or excessive cardiac lymphatic remodeling and insufficient lymph drainage have been implicated in several cardiovascular diseases including atherosclerosis and myocardial infarction (MI). Although the molecular mechanisms underlying the regulation of functional lymphatics are not fully understood, the interplay between lymphangiogenesis and immune regulation has recently been explored in relation to the initiation and development of these diseases. In this field, experimental therapeutic strategies targeting lymphangiogenesis have shown promise by reducing myocardial inflammation, edema and fibrosis, and improving cardiac function. On the other hand, however, whether lymphangiogenesis is beneficial or detrimental to cardiac transplant survival remains controversial. In the light of recent evidence, cardiac lymphangiogenesis, a thriving and challenging field has been summarized and discussed, which may improve our knowledge in the pathogenesis of cardiovascular diseases and transplant biology.

Morphological and biochemical changes of lymphatic vessels in the soleus muscle of mice after hindlimb unloading.

INTRODUCTION/AIMS: Lymphatic vessels are responsible for the removal of metabolic waste from body tissues. They also play a crucial role in skeletal muscle functioning thorough their high-energy metabolism. In this study we investigated whether disuse muscle atrophy induced by hindlimb unloading is associated with an alteration in the number of lymphatic vessels and differential expression of lymphangiogenic factors in the soleus muscle. METHODS: Male C57BL/6 mice were subjected to tail suspension (TS) for 2 or 4 weeks to induce soleus muscle atrophy. After TS, lymphatic and blood capillaries in the soleus muscle were visualized and counted by double staining with LYVE-1 and CD31. The protein and mRNA levels of vascular endothelial growth factor (VEGF)-C, VEGF-D, and vascular endothelial growth factor receptor-3 were measured by Western blotting and real-time reverse transcript polymerase chain reaction, respectively. RESULTS: TS for 2 weeks resulted in a significant decrease in the number of blood capillaries compared with controls. However, there was no significant change in the number of lymphatic capillaries. By contrast, TS for 4 weeks resulted in a significant decrease in the number of lymphatic and blood capillaries. We observed a significant decrease in the mRNA levels of VEGF-C and VEGF-D in mice subjected to TS for 4 weeks. DISCUSSION: The decrease of intramuscular lymphatic vessels may a crucial role in the process of muscle atrophy.

Avian Reticuloendotheliosis Viral Oncogene Related B Regulates Lymphatic Endothelial Cells during Vessel Maturation and Is Required for Lymphatic Vessel Function in Adult Mice.

NF-κB signals through canonical transcription factor p65 (RelA)/p50 and noncanonical avian reticuloendotheliosis viral oncogene related B (RelB)/p52 pathways. The RelA/p50 is involved in basal and inflammatory lymphangiogenesis. However, the role of RelB/p52 in lymphatic vessel biology is unknown. Herein, we investigated changes in lymphatic vessels (LVs) in mice deficient in noncanonical NF-κB signaling and the function of RelB in lymphatic endothelial cells (LECs). LVs were examined in Relb-/-, p52-/-, or control mice, and the gene expression profiles in LECs with RelB knockdown. Relb-/-, but not p52-/-, mice exhibited multiple LV abnormalities. They include the following: i) increased capillary vessel diameter, ii) reduced smooth muscle cell (SMC) coverage of mature vessels, iii) leakage, and iv) loss of active and passive lymphatic flow. Relb-/- mature LVs had thinner vessel walls, more apoptotic LECs and SMCs, and fewer LEC junctions. RelB knockdown LECs had decreased growth, survival, and adhesion, and dysregulated signaling pathways involving these cellular events. These results suggest that Relb-/- mice have abnormal LVs, mainly in mature vessels with reduced SMC coverage, leakage, and loss of contractions. RelB knockdown in LECs leads to reduced growth, survival, and adhesion. RelB plays a vital role in LEC-mediated LV maturation and function.

Role of simvastatin in tumor lymphangiogenesis and lymph node metastasis.

Lymphangiogenesis plays a crucial role in promoting cancer metastasis to sentinel lymph nodes (LNs) and beyond. Increasing data have shown that simvastatin, a cholesterol-lowering medication for the prevention of cardiovascular diseases, is involved in tumor growth and dissemination, and endothelial functions. This study aimed to investigate the potential effect of simvastatin on lymphatic formation and LN metastasis. Tumor models were established by subcutaneous injection of B16-F10 melanoma cells into mouse hind footpads. Simvastatin was administered (0.2 µg/g, intraperitoneal injection, IP) every other day for a total of eight times. Tissue samples were removed and examined by immunohistochemical staining and reverse transcription-polymerase chain reaction (RT-PCR) techniques. The lymphatics of LN, skin, liver, and lung exhibited morphological changes, and LN weight and metastatic area of the tumor group treated with simvastatin was lower than that of the untreated tumor group. Analysis of lymphatic size, area fraction, and lymphatic vessel density showed tissue specificity and variation to melanoma carcinogenesis in the simvastatin-treated group compared with the untreated group. In addition, LNs and cutaneous tissues showed altered expression of lymphangiogenic factors and inflammatory cytokines such as VEGF-A/-C/-D and TNF-α. These findings indicated that simvastatin may modify lymphangiogenesis and tumor progression in malignant melanoma.

Recent advances and new insights into muscular lymphangiogenesis in health and disease.

The lymphatic vessels have been implicated in maintenance of interstitial fluid homeostasis and immune responses, and pathological conditions including inflammation, wound healing, lymphedema and tumor progression. The knowledge about the lymphatic structure and function in muscles, especially in muscular disorders, remains fragmentary and elusive. LYVE-1-positive initial lymphatics are generally found around skeletal muscle fibers, but not distributed in a fiber type-specific manner. Recent advances in lymphatic research have identified that exercise stimuli trigger adaptive changes in the behavior of initial lymphatics and upregulate lymphangiogenesis. Increasing evidence has supported that muscle disorders are closely correlated with lymphatic dysfunction, growth and remodeling. During these processes, VEGFR-3 and its ligands VEGF-C and VEGF-D are important regulators of muscular lymphangiogenesis. Further studies are necessary to clarify the structural and molecular plasticity of mammalian muscular lymphatics in response to endurance training and pathological events.

Supermolecular drug challenge to overcome drug resistance in cancer cells.

Overcoming multidrug resistance (MDR) of cancer cells can be accomplished using drug delivery systems in large-molecular-weight ATP-binding cassette transporters before entry into phagolysosomes and by particle-cell-surface interactions. However, these hypotheses do not address the intratumoral heterogeneity in cancer. Anti-MDR must be related to alterations of drug targets, expression of detoxification, as well as altered proliferation. In this study, it is shown that the excellent efficacy and sustainability of anti-MDR is due to a stable ES complex because of the allosteric facilities of artificial enzymes when they are used as supermolecular complexes. The allosteric effect of supermolecular drugs can be explained by the induced-fit model and can provide stable feedback control systems through the loop transfer function of the Hill equation.

Brief Report: Treatment of Tumor Necrosis Factor-Transgenic Mice With Anti-Tumor Necrosis Factor Restores Lymphatic Contractions, Repairs Lymphatic Vessels, and May Increase Monocyte/Macrophage Egress.

OBJECTIVE: Recent studies have demonstrated that there is an inverse relationship between lymphatic egress and inflammatory arthritis in affected joints. As a model, tumor necrosis factor (TNF)-transgenic mice develop advanced arthritis following draining lymph node (LN) collapse, and loss of lymphatic contractions downstream of inflamed joints. It is unknown if these lymphatic deficits are reversible. This study was undertaken to test the hypothesis that anti-TNF therapy reduces advanced erosive inflammatory arthritis, associated with restoration of lymphatic contractions, repair of damaged lymphatic vessels, and evidence of increased monocyte egress. METHODS: TNF-transgenic mice with advanced arthritis and collapsed popliteal LNs were treated with anti-TNF monoclonal antibody (10 mg/kg weekly) or placebo for 6 weeks, and effects on knee synovitis, lymphatic vessel ultrastructure and function, and popliteal LN cellularity were assessed by ultrasound, histology, transmission electron microscopy (TEM), near-infrared indocyanine green imaging, and flow cytometry. RESULTS: Anti-TNF therapy significantly decreased synovitis (∼5-fold; P < 0.05 versus placebo), restored lymphatic contractions, and significantly increased the number of popliteal LN monocyte/macrophages (∼2-fold; P < 0.05 versus placebo). TEM demonstrated large activated macrophages attached to damaged lymphatic endothelium in mice with early arthritis, extensively damaged lymphatic vessels in placebo-treated mice with advanced arthritis, and rolling leukocytes in repaired lymphatic vessels in mice responsive to anti-TNF therapy. CONCLUSION: These findings support the concept that anti-TNF therapy ameliorates erosive inflammatory arthritis, in part via restoration of lymphatic vessel contractions and potential enhancement of inflammatory cell egress.

Lymph Nodes and Cancer Metastasis: New Perspectives on the Role of Intranodal Lymphatic Sinuses.

The lymphatic system is essential for transporting interstitial fluid, soluble antigen, and immune cells from peripheral tissues to lymph nodes (LNs). Functional integrity of LNs is dependent on intact lymphatics and effective lymph drainage. Molecular mechanisms that facilitate interactions between tumor cells and lymphatic endothelial cells (LECs) during tumor progression still remain to be identified. The cellular and molecular structures of LNs are optimized to trigger a rapid and efficient immune response, and to participate in the process of tumor metastasis by stimulating lymphangiogenesis and establishing a premetastatic niche in LNs. Several molecules, e.g., S1P, CCR7-CCL19/CCL21, CXCL12/CXCR4, IL-7, IFN-γ, TGF-ß, and integrin α4ß1 play an important role in controlling the activity of LN stromal cells including LECs, fibroblastic reticular cells (FRCs) and follicular dendritic cells (DCs). The functional stromal cells are critical for reconstruction and remodeling of the LN that creates a unique microenvironment of tumor cells and LECs for cancer metastasis. LN metastasis is a major determinant for the prognosis of most human cancers and clinical management. Ongoing work to elucidate the function and molecular regulation of LN lymphatic sinuses will provide insight into cancer development mechanisms and improve therapeutic approaches for human malignancy.

Medicinal facilities to B16F10 melanoma cells for distant metastasis control with a supramolecular complex by DEAE-dextran-MMA copolymer/paclitaxel.

The resistance of cancer cells to chemotherapeutic drugs (MDR) is a major problem to be solved. A supramolecular DEAE-dextran-MMA copolymer (DDMC)/paclitaxel (PTX) complex was obtained by using PTX as the guest and DDMC as the host having 50-300 nm in diameter. The drug resistance of B16F10 melanoma cells to paclitaxel was observed, but there is no drug resistance of melanoma cells to the DDMC/PTX complex in vitro. The cell death rate was determined using Michaelis-Menten kinetics, as the DDMC/PTX complex promoted allosteric supramolecular reaction to tubulin. The DDMC/PTX complex showed a very superior anti-cancer activity to paclitaxel alone in vivo. The median survival time (MST) of the saline, PTX, DDMC/PTX4 (particle size, 50 nm), and DDMC/PTX5 (particle size, 290 nm) groups were 120 h (T/C, 1.0), 176 h (T/C, 1.46), 328 h (T/C, 2.73), and 280 h (T/C, 2.33), respectively. The supramolecular DDMC/PTX complex showed the twofold effectiveness of PTX alone (p < 0.036). Histochemical analysis indicated that the administration of DDMC/PTX complex decreased distant metastasis and increased the survival of mice. A mouse of DDMC/PTX4 group in vivo was almost curing after small dermatorrhagia owing to its anti-angiogenesis, and it will be the hemorrhagic necrotic symptom of tumor by the release of "tumor necrosis factor alpha (TNF-α)" cytokine. As the result, the medicinal action of the DDMC/PTX complex will suppress the tumor-associated action of M2 macrophages and will control the metastasis of cancer cells.

Rapamycin inhibition of CFA-induced lymphangiogenesis in PLN is independent of mast cells.

Elucidation of the events responsible for the interaction between lymphatic endothelial cells (LECs) and mast cells (MCs) may prove to be a valuable source for controlling lymphangiogenesis. In the present study, we compared immunohistochemical and RT-PCR findings of the popliteal lymph node (PLN) and footpad skin in C57BL/6J and WBB6F1 mice, the MC-deficient strain. The results indicated that MCs play certain role in complete Freund's adjuvant-induced intranodal lymphangiogenesis. VEGF-A, VEGFR-2 and TNF-α were crucial factors in lymphangiogenesis both in the PLN and skin. Moreover, the in vivo administration of the specific mTOR inhibitor, rapamycin inhibited lymphangiogenesis independent of MCs in PLN rather than in the skin. Further study on anti-lymphangiogenic effect will contribute to our understanding of LEC and MC modulation in pathological lymphangiogenesis.

Anticancer efficacy of a supramolecular complex of a 2-diethylaminoethyl-dextran-MMA graft copolymer and paclitaxel used as an artificial enzyme.

The anticancer efficacy of a supramolecular complex that was used as an artificial enzyme against multi-drug-resistant cancer cells was confirmed. A complex of diethylaminoethyl-dextran-methacrylic acid methylester copolymer (DDMC)/paclitaxel (PTX), obtained with PTX as the guest and DDMC as the host, formed a nanoparticle 50-300 nm in size. This complex is considered to be useful as a drug delivery system (DDS) for anticancer compounds since it formed a stable polymeric micelle in water. The resistance of B16F10 melanoma cells to PTX was shown clearly through a maximum survival curve. Conversely, the DDMC/PTX complex showed a superior anticancer efficacy and cell killing rate, as determined through a Michaelis-Menten-type equation, which may promote an allosteric supramolecular reaction to tubulin, in the same manner as an enzymatic reaction. The DDMC/PTX complex showed significantly higher anticancer activity compared to PTX alone in mouse skin in vivo. The median survival times of the saline, PTX, DDMC/PTX4 (particle size 50 nm), and DDMC/PTX5 (particle size 290 nm) groups were 120 h (treatment (T)/control (C), 1.0), 176 h (T/C, 1.46), 328 h (T/C, 2.73), and 280 h (T/C, 2.33), respectively. The supramolecular DDMC/PTX complex showed twice the effectiveness of PTX alone (p < 0.036). Above all, the DDMC/PTX complex is not degraded in cells and acts as an intact supramolecular assembly, which adds a new species to the range of DDS.

Hypoxia and lymphangiogenesis in tumor microenvironment and metastasis.

Hypoxia and lymphangiogenesis are closely related processes that play a pivotal role in tumor invasion and metastasis. Intratumoral hypoxia is exacerbated as a result of oxygen consumption by rapidly proliferating tumor cells, insufficient blood supply and poor lymph drainage. Hypoxia induces functional responses in lymphatic endothelial cells (LECs), including cell proliferation and migration. Multiple factors (e.g., ET-1, AP-1, C/EBP-δ, EGR-1, NF-κB, and MIF) are involved in the events of hypoxia-induced lymphangiogenesis. Among them, HIF-1α is known to be the master regulator of cellular oxygen homeostasis, mediating transcriptional activation of lymphangiogenesis via regulation of signaling cascades like VEGF-A/-C/-D, TGF-ß and Prox-1 in experimental and human tumors. Although the underlying molecular mechanisms remain incompletely elucidated, the investigation of lymphangiogenesis in hypoxic conditions may provide insight into potential therapeutic targets for lymphatic metastasis.

Macrophages are important mediators of either tumor- or inflammation-induced lymphangiogenesis.

The lymphatic system provides important functions for tissue fluid homeostasis and immune response. Lymphangiogenesis, the formation of new lymphatics, comprises a series of complex cellular events in vitro or in vivo, e.g., proliferation, differentiation, and sprouting. Recent evidence has implied that macrophages act as a direct structural contributor to lymphatic endothelial walls or secret VEGF-C/-D and VEGF-A to initiate lymphangiogenesis in inflamed or tumor tissues. Bone marrow-derived macrophages are versatile cells that express different functional programs in response to exposure to microenvironmental signals, and can be identified by specific expression of a number of proteins, F4/80, CD11b, and CD68. Several causative factors, e.g., NF-κB, IL-1ß, TNF-α, SDF-1, M-CSF, especially TonEBP/VEGF-C signaling, may be actively involved in macrophage-induced lymphangiogenesis. Alteration of macrophage phenotype and function has a profound effect on the development and progression of inflammation and malignancy, and macrophage depletion for controlling lymphangiogenesis may provide a novel approach for prevention and treatment of lymphatic-associated diseases.

Multiple expressions of lymphatic markers and morphological evolution of newly formed lymphatics in lymphangioma and lymph node lymphangiogenesis.

The rapid evolution of reliable technology combined with increasing number of specific markers for lymphatic endothelial cells (LECs) facilitates the investigation of lymphangiogenesis in developing and diseased tissues. Here, we injected incomplete Freund's adjuvant (IFA) peritoneally into BALB/c and nonobese diabetic (NOD) mice to induce lymphangioma and found atypical lymphatic accumulations with intervening fibrous tissue and lymphoid aggregates. Lymphatic markers, LYVE-1 and podoplanin, were used to specifically define the morphological features of the neoplastic lymphatics. The NOD mice (affected by an autoimmune disorder) had fewer and smaller lymphangiomas than the BALB/c mice. Injection of IFA in the footpad skin of the mice also disturbed draining regional lymph node lymphangiogenesis and caused enlargement of popliteal lymph nodes. Molecular analyses of the LECs indicated potential interventions for lymphangioma through vascular endothelial growth factor (VEGF)-A/-C/-D and their receptors, VEGF receptors-2/-3, and Prox-1 signaling pathways. These findings represent an important link between multiple factors and lymphatic disorders.

Inhibition of lymphangiogenesis and lymphatic drainage via vascular endothelial growth factor receptor 3 blockade increases the severity of inflammation in a mouse model of chronic inflammatory arthritis.

OBJECTIVE: This study was undertaken to investigate the effect of lymphatic inhibition on joint and draining lymph node (LN) pathology during the course of arthritis progression in mice. METHODS: Tumor necrosis factor (TNF)-transgenic mice were used as a model of chronic inflammatory arthritis. Mice were subjected to contrast-enhanced magnetic resonance imaging to obtain ankle and knee joint synovial volumes and draining popliteal LN volumes before and after 8 weeks of treatment with vascular endothelial growth factor receptor 3 (VEGFR-3) neutralizing antibody, VEGFR-2 neutralizing antibody, or isotype IgG. Animals were subjected to near-infrared lymphatic imaging to determine the effect of VEGFR-3 neutralization on lymph transport from paws to draining popliteal LNs. Histologic, immunohistochemical, and reverse transcriptase-polymerase chain reaction analyses were used to examine lymphatic vessel formation and the morphology of joints and popliteal LNs. RESULTS: Compared with IgG treatment, VEGFR-3 neutralizing antibody treatment significantly decreased the size of popliteal LNs, the number of lymphatic vessels in joints and popliteal LNs, lymphatic drainage from paws to popliteal LNs, and the number of VEGF-C-expressing CD11b+ myeloid cells in popliteal LNs. However, it increased the synovial volume and area of inflammation in ankle and knee joints. VEGFR-2 neutralizing antibody, in contrast, inhibited both lymphangiogenesis and joint inflammation. CONCLUSION: These findings indicate that lymphangiogenesis and lymphatic drainage are reciprocally related to the severity of joint lesions during the development of chronic arthritis. Lymphatic drainage plays a beneficial role in controlling the progression of chronic inflammation.

Lymphatic endothelial cells, lymphedematous lymphangiogenesis, and molecular control of edema formation.

Lymphedema, defined as the abnormal accumulation of protein-rich fluid in soft tissues, results from the dysfunction of lymphatic system, an imbalance between lymph formation and its absorption into the initial lymphatics. Primary lymphedema occurs rarely on idiopathic or developmental abnormalities, especially hypoplasia or aplasia of lymphatics. Secondary lymphedema commonly develops when lymph transport is impaired due to lymphatic damage or resection of lymph nodes in surgery, infection, and radiation. Lymphatic endothelial cells (LECs) actively participate in the phenotypic consequences of a deranged lymphangiogenesis relating to tissue fluid accumulation in the pathogenesis of lymphedema. Recent insights into molecular genetic bases have shown an updated genotype-phenotype correlation between lymphangiogenesis, lymphatic function, and lymphedema. FOXC2, EphrinB2, VEGFR-3, VEGF-C, angiopoietin-2, Prox-1 and podoplanin have proved to be important factors of the genetic cascade linking to hereditary lymphedema, and embryonic and postnatal lymphatic development. FOXC2 may have a key role in regulating interactions between LECs and smooth muscle cells, and in the morphogenesis of lymphatic valves. Reduced VEGFR-3 tyrosine kinase activity and subsequent failure in transducing sufficient physiological VEGF-C/-D signals may affect LEC function and structure in the intercellular junctions and peri-lymphatic components. Identification of genetic markers in humans and animal models would facilitate the management of environmental factors influencing the expression and severity of lymphedema, and provide a basis for developing novel targeted therapies for the disease.

Lymphangiogenesis, myeloid cells and inflammation.

The lymphatic system is essential for the maintenance of tissue fluid balance, immune surveillance and the absorption of fatty acids in the gastrointestinal tract. The lymphatic circulation is also a key player in disease processes such as cancer metastasis, lymphedema and various inflammatory disorders. With the identification of specific growth factors for lymphatic endothelial cells and markers that distinguish blood and lymphatic vessels, as well as the development of in vivo imaging technologies that provide new tools to examine the lymphatic drainage function in real time, many advancements have been made in lymphatic vascular research during the past few years. Despite these significant achievements, our understanding of the role of lymphatics in disease processes other than cancer metastasis is still rather limited. The current review will focus on the recent progress made in studies of lymphatics in inflammatory disorders.

Lymphatic vascular endothelial hyaluronan receptor (LYVE)-1- and CCL21-positive lymphatic compartments in the diabetic thymus.

To explore the biological significance of the lymphatics in the autoimmune process, the thymus from non-obese diabetic (NOD) mice was evaluated by histochemistry and western blot analysis. Thymic lymphatic endothelial cells showed suggestive expression patterns of the functional molecules lymphatic vascular endothelial hyaluronan receptor (LYVE)-1, CCL21, CD31 and podoplanin. With increasing age, the expression of CCL21 was reduced in the medullary epithelial cells and lymphatics. Of note, LYVE-1-expressing lymphatics, filled with a cluster of thymocytes, increased in number and size and extended from the corticomedullary boundary into the medulla as the insulitis progressed. The development of lymphatic compartments was occasionally accompanied by a regional disappearance between the cortex and medulla. The CD4- and CD8-positive T cells frequently penetrated through the slender lymphatic walls. The epithelial reticular cell layer lining the perivascular spaces was extensively stained with cytokeratin, but the expression of cytokeratin showed an age-dependent decrease. These findings indicate that the occurrence of LYVE-1-expressing lymphatic compartments and the alteration of CCL21 expression in the lymphatics may be involved in defective thymocyte differentiation and migration, and play a significant role in insulitic and diabetic processes.

Lymphatic endothelial cells, tumor lymphangiogenesis and metastasis: New insights into intratumoral and peritumoral lymphatics.

Lymphatic metastasis of tumor cells represents a series of extremely complex and sequential processes that include dissemination and invasion into surrounding stromal tissues from primary tumors, penetration into lymphatic walls and implantation in regional lymph nodes, and extravasation or proliferation in parenchyma of target organs. Recent developments in lymphatic biology and research, especially the application of unique molecular markers specific for lymphatic endothelial cells (LECs), LYVE-1, Prox-1 and podoplanin have provided exciting new insights into the tumor microenvironment and LEC-tumor cell interface. To date, established factors for determining the behavior and prognosis of primary tumors have been emphasized morphologically and physiologically, i.e., lymphatic impairment and vessel density, dysfunction of lymphatic valves, interstitial fluid pressure, as well as a series of lymphangiogenic growth factors including VEGF-C/-D, and other cytokines and chemokines. Increasing knowledge of the tumor biological significance in lymphatics within the tumors (intratumoral lymphatics, ITLs) and at the tumor periphery (peritumoral lymphatics, PTLs) has greatly promoted understanding of tumor access into the lymphatic system by inducing lymphangiogenesis or by co-opting preexisting lymphatics. Therefore, the targeting PTLs and ITLs, which have been proposed as an important route for antimetastatic approach, are deemed worthy of further study in various animal tumor models and human tumors.