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Lymphangiogenesis and Lung Cancer

  • Ana Rouzaut
    Correspondence
    Address for correspondence: Ana Rouzaut, Center for Applied Medical Research, University of Navarra. Av. Pío XII 55, 31008 Pamplona, Spain
    Affiliations
    Oncology Division, Centre for Applied Medical Research, University of Navarra, Pamplona, Spain

    Department of Biochemistry, School of Medicine, University of Navarra, Pamplona, Spain
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  • Marta Irigoyen
    Affiliations
    Oncology Division, Centre for Applied Medical Research, University of Navarra, Pamplona, Spain

    Department of Histology and Pathology, University of Navarra, Pamplona, Spain
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  • Luis M. Montuenga
    Affiliations
    Oncology Division, Centre for Applied Medical Research, University of Navarra, Pamplona, Spain

    Department of Histology and Pathology, University of Navarra, Pamplona, Spain
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      THE LYMPHATICS: NEW TOOLS AND NEW RESEARCH AVENUES

      The lymphatic vasculature maintains tissue homeostasis and sustains immune function by guiding leukocytes and activated antigen presenting cells toward the lymph nodes. The lymphatic system is also relevant in cancer progression because cancer cells frequently spread out of the original tumor through the lymphatic capillaries.
      • Saharinen P
      • Tammela T
      • Karkkainen MJ
      • Alitalo K
      Lymphatic vasculature: development, molecular regulation and role in tumor metastasis and inflammation.
      The detection of cancer cells in lymphatic vessels and regional lymph nodes is a key criterion in the staging of many human tumors, including lung cancer, and is used as decisive element for therapeutic intervention.
      • Tobler NE
      • Detmar M
      Tumor and lymph node lymphangiogenesis: impact on cancer metastasis.
      Although the clinical relevance of lymph node involvement is well known, the molecular mechanisms by which tumor cells spread via lymphatic vessels are less understood. In fact, the study of the lymphatic vasculature has long been hampered because of a lack of lymphatic-specific markers. Enormous progress has occurred in this field during the past decade through the identification of specific lymphatic endothelial cell markers such as VEGFR3, the receptor for vascular endothelial growth factors C and D, the hyaluronan receptor LYVE-1, or the mucin-like transmembrane protein podoplanin.
      • Alitalo K
      • Tammela T
      • Petrova TV
      Lymphangiogenesis in development and human disease.
      The availability of these markers has allowed the quantification of lymphatic vessel density in tumor biopsies and the isolation of lymphatic endothelial cells for in vitro studies.

      LYMPHANGIOGENESIS AND CANCER: MOLECULAR REGULATORY PATHWAYS

      Although the hemangiogenic activity of tumor cells was established decades ago,
      • Folkman J
      Angiogenesis.
      relatively recent data have shown that tumors are also able to induce lymphangiogenesis through the production of pro-lymphangiogenic factors such as VEGF-C.
      • Alitalo K
      • Tammela T
      • Petrova TV
      Lymphangiogenesis in development and human disease.
      What is more relevant, a great number of clinicopathological studies show good correlation between lymphatic vessel density in human tumors and poor prognosis.
      • Saharinen P
      • Tammela T
      • Karkkainen MJ
      • Alitalo K
      Lymphatic vasculature: development, molecular regulation and role in tumor metastasis and inflammation.
      In fact, consensus criteria for the immunohistochemical evaluation of lymphangiogenesis in solid tumors have been recently proposed (Figure 1).
      • Van der Auwera I
      • Cao Y
      • Tille JC
      • et al.
      First international consensus on the methodology of lymphangiogenesis quantification in solid human tumours.
      Figure thumbnail gr1
      FIGURE 1A, Endothelial growth factors and receptors involved in lymphatic endothelial cell growth signaling. B, Binding of VEGF-C/D to VEGFR3 leads to its phosphorylation and to the activation of proliferation, migration, and survival pathways. C, D, Immunohistochemical staining of lymphatic vessels using anti-podoplanin antibody in human lung adenocarcinoma samples. Left, Tumor marginal lymphatic vessels. Right, Adenocarcinoma cells within the lumen of a lymphatic vessel.
      The information gathered so far about the mechanisms involved in the regulation of lymphatic vessel growth shows that there are overlapping molecular players shared between hemangiogenesis and lymphangiogenesis. The main regulatory pathway specific for lymphatic endothelial cells is the axis formed by VEGFR-3 (also called Flt-4) and its ligands: VEGF-C and VEGFC-D. VEGFR3 was the first specific lymphatic endothelial cell marker discovered and shows high structural homology with VEGFR2, which is more directly involved in vascular angiogenesis.
      • Kaipainen A
      • Korhonen J
      • Mustonen T
      • et al.
      Expression of the fms-like tyrosine kinase 4 gene becomes restricted to lymphatic endothelium during development.
      VEGF-C and VEGF-D are secreted as immature pro-peptides and are proteolyzed by extracellular convertases.
      • Wissmann C
      • Detmar M
      Pathways targeting tumor lymphangiogenesis.
      Their processing increases their affinity for VEGFR3 and (less efficiently) for VEGFR2. Ligand binding induces VEGFR3 tyrosine auto-phosphorylation and the recruitment of intracellular signaling proteins, which promote lymphatic endothelial cell proliferation, migration, and survival.
      • Wissmann C
      • Detmar M
      Pathways targeting tumor lymphangiogenesis.
      Therefore, the expression and activation of the VEGFC-D/VEGFR3 pathway seem to be a key molecular link between cancer and lymphangiogenesis, making it a good target to prevent the metastatic spread of solid tumors. Nevertheless, newly published data suggest that the picture is far more complex. The identification of co-receptors for VEGF-C and VEGF-D such as neuropilin-2,
      • Yuan L
      • Moyon D
      • Pardanaud L
      • et al.
      Abnormal lymphatic vessel development in neuropilin 2 mutant mice.
      or the β1 and α9-integrins, suggests the existence of more intricate signaling pathways.
      • Vlahakis NE
      • Young BA
      • Atakilit A
      • Sheppard D
      The lymphangiogenic vascular endothelial growth factors VEGF-C and -D are ligands for the integrin alpha9beta1.
      Recent in vivo studies have shown that the hemangiogenic factor VEGF-A also induces lymphangiogenesis and promotes tumor metastasis.
      • 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.
      Furthermore, other growth factors such as angiopoietin, fibroblast growth factor 2, platelet-derived growth factor B, and hepatocyte growth factor
      • Cueni LN
      • Detmar M
      New insights into the molecular control of the lymphatic vascular system and its role in disease.
      stimulate lymphatic vessel growth, suggesting that we have only spotted the tip of a complicated regulatory process.
      Studies performed in animal models strongly suggest that tumor secreted VEGF-C is also capable of stimulating the motility and the ability of cancer cells to cross the lymphatic endothelial wall.
      • Su JL
      • Yang PC
      • Shih JY
      • et al.
      The VEGF-C/Flt-4 axis promotes invasion and metastasis of cancer cells.
      Nevertheless, there is still some degree of controversy regarding the relevance of either peritumoral and/or intratumoral lymphatic vessels in solid tumors. On one hand, several findings suggest that the functional lymphatics located in the tumor margin alone are sufficient for lymphatic metastasis.
      • Padera TP
      • Kadambi A
      • di Tomaso E
      • et al.
      Lymphatic metastasis in the absence of functional intratumor lymphatics.
      On the other hand, increasing experimental evidence shows the existence of intratumoral lymphatics and suggests their association with poor survival.
      • Ji RC
      Lymphatic endothelial cells, tumor lymphangiogenesis and metastasis: new insights into intratumoral and peritumoral lymphatics.
      The question of intra- versus peritumoral localization is interesting when evaluating tumors arising from organs, such as the lung, with heterogeneous lymphatic distribution. In the lung, the lymphatic vasculature extends as far as the bronchioles but fails to reach the alveoli.
      • Itoh H
      • Nakatsu M
      • Yoxtheimer LM
      • Uematsu H
      • Ohno Y
      • Hatabu H
      Structural basis for pulmonary functional imaging.

      LYMPHANGIOGENESIS IN LUNG CANCER

      Expression studies demonstrated that the levels of VEGF-C and VEGF-D mRNA and protein are elevated in small cell lung cancer cell lines.
      • Tanno S
      • Ohsaki Y
      • Nakanishi K
      • Toyoshima E
      • Kikuchi K
      Human small cell lung cancer cells express functional VEGF receptors, VEGFR-2 and VEGFR-3.
      Interestingly, the evaluation of VEGF-C, VEGF-D, and VEGFR3 expression in clinicopathological samples suggest that they are frequently overexpressed in non-small cell lung cancer (NSCLC) cells. This increase is associated with lower survival rates.
      • Renyi-Vamos F
      • Tovari J
      • Fillinger J
      • et al.
      Lymphangiogenesis correlates with lymph node metastasis, prognosis, and angiogenic phenotype in human non-small cell lung cancer.
      Moreover, serum VEGF-C determination in NSCLC has been used as a marker for lymph node metastasis, and its evaluation, along with computed tomographic examination, has been proposed as a complement for accurate lymph node staging.
      • Tamura M
      • Oda M
      • Tsunezuka Y
      • et al.
      Chest CT and serum vascular endothelial growth factor-C level to diagnose lymph node metastasis in patients with primary non-small cell lung cancer.
      Despite evidence on the presence of an active proliferating network of lymphatic vessels in the invading front of lung adenocarcinomas, a number of questions about lymphangiogenesis in lung cancer remains: among others, the presence of intratumoral lymphatics
      • Koukourakis MI
      • Giatromanolaki A
      • Sivridis E
      • et al.
      LYVE-1 immunohistochemical assessment of lymphangiogenesis in endometrial and lung cancer.
      or the existence of an association between its heterogeneous lymphatic support and the frequency of metastatic spread. Finally, it is not clear to what extent the relevance of lymphatic vasculature in NSCLC changes with histological type, tumor size, or location within the lung.

      LYMPHANGIOGENESIS INHIBITION

      Several approaches have been used for the therapeutic targeting of tumoral lymphatic growth in preclinical and clinical studies. Some of them used specific antibodies developed against VEGF-C, VEGF-D, or VEGFR3 to neutralize their activity.
      • Wissmann C
      • Detmar M
      Pathways targeting tumor lymphangiogenesis.
      Alternatively, soluble versions of VEGFR3 receptor have been developed and used in preclinical trials.
      • Wissmann C
      • Detmar M
      Pathways targeting tumor lymphangiogenesis.
      For example, the overexpression of soluble VEGFR-3 by lung cancer cells reduced tumoral lymphatic vessels and the incidence of metastasis to sentinel lymph nodes in animal models.
      • He Y
      • Kozaki K
      • Karpanen T
      • et al.
      Suppression of tumor lymphangiogenesis and lymph node metastasis by blocking vascular endothelial growth factor receptor 3 signaling.
      Besides, some small molecule inhibitors of VEGFR2 kinase activity that have also been shown to inhibit VEGFR3 kinase activity are currently being tested in clinical trials. Examples of these multiple inhibitors are BAY 43-9006 (sorafenib), PTK787/ZK222584 (Vatalanib), CEP-7055, and indolinones such as MAE87 and MAZ51.
      • Achen MG
      • McColl BK
      • Stacker SA
      Focus on lymphangiogenesis in tumor metastasis.
      The potential clinical benefit of anti-lymphangiogenesis molecular targeted approaches is promising. A combined therapy to block both hemangiogenesis and lymphangiogenesis may even be considered. Nevertheless, some potential adverse effects, mainly in hematopoiesis and tissue edema, are strong reasons to act cautiously when translating them into clinical practice.

      ACKNOWLEDGMENTS

      AR and LMM are supported by the UTE-CIMA project. This work was partially funded by EU Grant QLRT-2001-01735. We are very grateful to Drs. María J. Pajares and Jackeline Agorreta for the immunohistochemical pictures and to Dr. Ruben Pío for critical reading of the manuscript. We thank Mr. Heber Longas for the artwork included in this review.

      REFERENCES

        • Saharinen P
        • Tammela T
        • Karkkainen MJ
        • Alitalo K
        Lymphatic vasculature: development, molecular regulation and role in tumor metastasis and inflammation.
        Trends Immunol. 2004; 25: 387-395
        • Tobler NE
        • Detmar M
        Tumor and lymph node lymphangiogenesis: impact on cancer metastasis.
        J Leukoc Biol. 2006; 80: 691-696
        • Alitalo K
        • Tammela T
        • Petrova TV
        Lymphangiogenesis in development and human disease.
        Nature. 2005; 438: 946-953
        • Folkman J
        Angiogenesis.
        Annu Rev Med. 2006; 57: 1-18
        • Van der Auwera I
        • Cao Y
        • Tille JC
        • et al.
        First international consensus on the methodology of lymphangiogenesis quantification in solid human tumours.
        Br J Cancer. 2006; 95: 1611-1625
        • Kaipainen A
        • Korhonen J
        • Mustonen T
        • et al.
        Expression of the fms-like tyrosine kinase 4 gene becomes restricted to lymphatic endothelium during development.
        Proc Natl Acad Sci USA. 1995; 92: 3566-3570
        • Wissmann C
        • Detmar M
        Pathways targeting tumor lymphangiogenesis.
        Clin Cancer Res. 2006; 12: 6865-6868
        • Yuan L
        • Moyon D
        • Pardanaud L
        • et al.
        Abnormal lymphatic vessel development in neuropilin 2 mutant mice.
        Development. 2002; 129: 4797-4806
        • Vlahakis NE
        • Young BA
        • Atakilit A
        • Sheppard D
        The lymphangiogenic vascular endothelial growth factors VEGF-C and -D are ligands for the integrin alpha9beta1.
        J Biol Chem. 2005; 280: 4544-4552
        • 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: 1089-1099
        • Cueni LN
        • Detmar M
        New insights into the molecular control of the lymphatic vascular system and its role in disease.
        J Invest Dermatol. 2006; 126: 2167-2177
        • Su JL
        • Yang PC
        • Shih JY
        • et al.
        The VEGF-C/Flt-4 axis promotes invasion and metastasis of cancer cells.
        Cancer Cell. 2006; 9: 209-223
        • Padera TP
        • Kadambi A
        • di Tomaso E
        • et al.
        Lymphatic metastasis in the absence of functional intratumor lymphatics.
        Science. 2002; 296: 1883-1886
        • Ji RC
        Lymphatic endothelial cells, tumor lymphangiogenesis and metastasis: new insights into intratumoral and peritumoral lymphatics.
        Cancer Metast Rev. 2006; 25: 677-694
        • Itoh H
        • Nakatsu M
        • Yoxtheimer LM
        • Uematsu H
        • Ohno Y
        • Hatabu H
        Structural basis for pulmonary functional imaging.
        Eur J Radiol. 2001; 37: 143-154
        • Tanno S
        • Ohsaki Y
        • Nakanishi K
        • Toyoshima E
        • Kikuchi K
        Human small cell lung cancer cells express functional VEGF receptors, VEGFR-2 and VEGFR-3.
        Lung Cancer. 2004; 46: 11-19
        • Renyi-Vamos F
        • Tovari J
        • Fillinger J
        • et al.
        Lymphangiogenesis correlates with lymph node metastasis, prognosis, and angiogenic phenotype in human non-small cell lung cancer.
        Clin Cancer Res. 2005; 11: 7344-7353
        • Tamura M
        • Oda M
        • Tsunezuka Y
        • et al.
        Chest CT and serum vascular endothelial growth factor-C level to diagnose lymph node metastasis in patients with primary non-small cell lung cancer.
        Chest. 2004; 126: 342-346
        • Koukourakis MI
        • Giatromanolaki A
        • Sivridis E
        • et al.
        LYVE-1 immunohistochemical assessment of lymphangiogenesis in endometrial and lung cancer.
        J Clin Pathol. 2005; 58: 202-206
        • Wissmann C
        • Detmar M
        Pathways targeting tumor lymphangiogenesis.
        Clin Cancer Res. 2006; 12: 6865-6868
        • He Y
        • Kozaki K
        • Karpanen T
        • et al.
        Suppression of tumor lymphangiogenesis and lymph node metastasis by blocking vascular endothelial growth factor receptor 3 signaling.
        J Natl Cancer Inst. 2002; 94: 819-825
        • Achen MG
        • McColl BK
        • Stacker SA
        Focus on lymphangiogenesis in tumor metastasis.
        Cancer Cell. 2005; 7: 121-127