Abstract
Tumor metastasis to lymph nodes is a key indicator of patient survival, and is enhanced by the neo-lymphatics induced by tumor-secreted VEGF-C or VEGF-D, acting via VEGFR-3 signalling. These targets constitute important avenues for anti-metastatic treatment. Despite this new understanding, clinical observations linking metastasis with tumor depth or location suggest that lymphangiogenic growth factors are not the sole determinants of metastasis. Here we explored the influence of tumor proximity to lymphatics capable of responding to growth factors on nodal metastasis in a murine VEGF-D over-expression tumor model. We found that primary tumor location profoundly influenced VEGF-D-mediated lymph node metastasis: 89 % of tumors associated with the flank skin metastasised, in contrast with only 19 % of tumors located more deeply on the body wall (p < 0.01). Lymphatics in metastatic tumors arose from small lymphatics, and displayed distinct molecular and morphological profiles compared with those found in normal lymphatics. Smaller lymphatic subtypes were more abundant in skin (2.5-fold, p < 0.01) than in body wall, providing a richer source of lymphatics for VEGF-D+ skin tumors, a phenomenon also confirmed in human samples. This study shows that the proximity of a VEGF-D+ primary tumor to small lymphatics is an important determinant of metastasis. These observations may explain why tumor location relative to the lymphatic network is prognostically important for some human cancers.
Similar content being viewed by others
Abbreviations
- BW:
-
Body wall
- DAB:
-
Diaminobenzidene
- IL:
-
Initial lymphatics
- LEC:
-
Lymphatic endothelial cell
- LN:
-
Lymph node
- LVD:
-
Lymphatic vessel density
- LYVE:
-
Lymphatic vessel endothelial hyaluronan receptor
- Np:
-
Neuropilin
- PC:
-
Pre-collector
- PECAM-1:
-
Platelet endothelial cell adhesion molecule-1
- SCID/NOD:
-
Severe combined immunodeficiency/non-obese diabetic mice
- SK:
-
Skin
- SMC:
-
Smooth muscle cell
- VEGF:
-
Vascular endothelial growth factor
References
Markovic SN, Erickson LA, Rao RD et al (2007) Malignant melanoma in the 21st century, part 2: staging, prognosis, and treatment. Mayo Clin Proc 82:490–513
DeVita VT, Hellman S, Rosenberg A (2001) Cancer, principles and practice of oncology, 6th edn. Lippincott, Williams and Wilkins, Philadelphia, PA
Mandriota SJ, Jussila L, Jeltsch M et al (2001) Vascular endothelial growth factor-C-mediated lymphangiogenesis promotes tumour metastasis. EMBO J 20:672–682
Skobe M, Hawighorst T, Jackson DG et al (2001) Induction of tumor lymphangiogenesis by VEGF-C promotes breast cancer metastasis. Nat Med 7:192–198
Stacker SA, Caesar C, Baldwin ME et al (2001) VEGF-D promotes the metastatic spread of tumor cells via the lymphatics. Nat Med 7:186–191
Nagy JA, Vasile E, Feng D et al (2002) Vascular permeability factor/vascular endothelial growth factor induces lymphangiogenesis as well as angiogenesis. J Exp Med 196:1497–1506
Schietroma C, Cianfarani F, Lacal PM et al (2003) Vascular endothelial growth factor-C expression correlates with lymph node localization of human melanoma metastases. Cancer 98:789–797
White JD, Hewett PW, Kosuge D et al (2002) Vascular endothelial growth factor-D expression is an independent prognostic marker for survival in colorectal carcinoma. Cancer Res 62:1669–1675
Stacker SA, Williams RA, Achen MG (2004) Lymphangiogenic growth factors as markers of tumor metastasis. APMIS 112:539–549
Caunt M, Mak J, Liang WC et al (2008) Blocking neuropilin-2 function inhibits tumor cell metastasis. Cancer Cell 13:331–342
Achen MG, Mann GB, Stacker SA (2006) Targeting lymphangiogenesis to prevent tumour metastasis. Br J Cancer 94:1355–1360
Achen MG, McColl BK, Stacker SA (2005) Focus on lymphangiogenesis in tumor metastasis. Cancer Cell 7:121–127
Shayan R, Achen MG, Stacker SA (2006) Lymphatic vessels in cancer metastasis: bridging the gaps. Carcinogenesis 27:1729–1738
Goydos JS, Gorski DH (2003) Vascular endothelial growth factor C mRNA expression correlates with stage of progression in patients with melanoma. Clin Cancer Res 9:5962–5967
Nakamura Y, Yasuoka H, Tsujimoto M et al (2005) Lymph vessel density correlates with nodal status, VEGF-C expression, and prognosis in breast cancer. Breast Cancer Res Treat 91:125–132
Shayan R, Karnezis T, Murali R et al (2012) Variations in tumor lymphatic density in primary cutaneous melanomas predicts risk of lymph node metastasis. Histopathology. doi:10.1111/j.1365-2559.2012.04310.x.
Stacker SA, Achen MG, Jussila L et al (2002) Lymphangiogenesis and cancer metastasis. Nat Rev Cancer 2:573–583
Alitalo K, Tammela T, Petrova TV (2005) Lymphangiogenesis in development and human disease. Nature (Lond) 438:946–953
Achen MG, Stacker SA (2008) Molecular control of lymphatic metastasis. Ann NY Acad Sci 1131:225–234
Debinski W, Slagle-Webb B, Achen MG et al (2001) VEGF-D is an X-linked/AP-1 regulated putative onco-angiogen in human glioblastoma multiforme. Mol Med 7:598–608
Cunningham JE, Juri AL, Oman L et al (2006) Is risk of axillary lymph node metastasis associated with proximity of breast cancer to the skin? Breast Cancer Res Treat 100:319–328
Onogawa S, Kitadai Y, Tanaka S et al (2004) Expression of VEGF-C and VEGF-D at the invasive edge correlates with lymph node metastasis and prognosis of patients with colorectal carcinoma. Cancer Sci 95:32–39
Bevacqua SJ, Welch DR, Diez de Pinos SM et al (1990) Quantitation of human melanoma, carcinoma and sarcoma tumor cell adhesion to lymphatic endothelium. Lymphology 23:4–14
Padera TP, Kadambi A, di Tomaso E et al (2002) Lymphatic metastasis in the absence of functional intratumor lymphatics. Science 296:1883–1886
Smith KJ, Jones PF, Burke DA et al (2011) Lymphatic vessel distribution in the mucosa and submucosa and potential implications for T1 colorectal tumors. Dis Colon Rectum 54:35–40
Scavelli C, Weber E, Agliano M et al (2004) Lymphatics at the crossroads of angiogenesis and lymphangiogenesis. J Anat 204:433–449
Van der Auwera I, Cao Y, Tille JC et al (2006) First international consensus on the methodology of lymphangiogenesis quantification in solid human tumours. Br J Cancer 95:1611–1625
Pepper MS, Skobe M (2003) Lymphatic endothelium: morphological, molecular and functional properties. J Cell Biol 163:209–213
Makinen T, Adams RH, Bailey J et al (2005) PDZ interaction site in ephrinB2 is required for the remodeling of lymphatic vasculature. Genes Dev 19:397–410
Baldwin ME, Stacker SA, Achen MG (2002) Molecular control of lymphangiogenesis. BioEssays 24:1030–1040
Adams RH, Alitalo K (2007) Molecular regulation of angiogenesis and lymphangiogenesis. Nat Rev Mol Cell Biol 8:464–478
Tammela T, Saaristo A, Holopainen T et al (2007) Therapeutic differentiation and maturation of lymphatic vessels after lymph node dissection and transplantation. Nat Med 13:1458–1466
Karnezis T, Shayan R, Caesar C et al (2012) VEGF-D promotes tumor metastasis by regulating prostaglandins produced by the collecting lymphatic endothelium. Cancer Cell 21:181–195
Azzali G (2007) Tumor cell transendothelial passage in the absorbing lymphatic vessel of transgenic adenocarcinoma mouse prostate. Am J Pathol 170:334–346
He Y, Rajantie I, Pajusola K et al (2005) Vascular endothelial cell growth factor receptor 3-mediated activation of lymphatic endothelium is crucial for tumor cell entry and spread via lymphatic vessels. Cancer Res 65:4739–4746
Hoshida T, Isaka N, Hagendoorn J et al (2006) Imaging steps of lymphatic metastasis reveals that vascular endothelial growth factor-C increases metastasis by increasing delivery of cancer cells to lymph nodes: therapeutic implications. Cancer Res 66:8065–8075
Shayan R, Karnezis T, Tsantikos E et al (2007) A system for quantifying the patterning of the lymphatic vasculature. Growth Factors 25:417–425
Achen MG, Jeltsch M, Kukk E et al (1998) Vascular endothelial growth factor D (VEGF-D) is a ligand for the tyrosine kinases VEGF receptor 2 (Flk1) and VEGF receptor 3 (Flt4). Proc Natl Acad Sci USA 95:548–553
Stacker SA, Stenvers K, Caesar C et al (1999) Biosynthesis of vascular endothelial growth factor-D involves proteolytic processing which generates non-covalent homodimers. J Biol Chem 274:32127–32136
Saaristo A, Tammela T, Farkkila A et al (2006) Vascular endothelial growth factor-C accelerates diabetic wound healing. Am J Pathol 169:1080–1087
Muthuchamy M, Gashev A, Boswell N et al (2003) Molecular and functional analyses of the contractile apparatus in lymphatic muscle. FASEB J 17:920–922
Karpanen T, Egeblad M, Karkkainen MJ et al (2001) Vascular endothelial growth factor C promotes tumor lymphangiogenesis and intralymphatic tumor growth. Cancer Res 61:1786–1790
Wirzenius M, Tammela T, Uutela M et al (2007) Distinct vascular endothelial growth factor signals for lymphatic vessel enlargement and sprouting. J Exp Med 204:1431–1440
Farnsworth RH, Karnezis T, Shayan R et al (2011) A role for bone morphogenic protein-4 in vascular endothelial growth factor-D mediated tumor growth, metastasis and vessel remodelling. Cancer Res 71:6547–6557
Achen MG, Stacker SA (2012) Vascular endothelial growth factor-D: signalling mechanisms, biology and clinical relevance. Growth Factors 30:283–296
Shayan R, Rozen W, Bernard S et al (2008) Perforator dilatation induced by body weight gain is not reversed by subsequent weight loss: implications for perforator flaps. Plast Reconstr Surg 122:1765–1772
Stadelmann WK, Reintgen DS (1998) Prognosis in malignant melanoma. Hematol Oncol Clin North Am 12:767–796, vi
Deutsch A, Lubach D, Nissen S et al (1992) Ultrastructural studies on the invasion of melanomas in initial lymphatics of human skin. J Invest Dermatol 98:64–67
Gordon EJ, Rao S, Pollard JW et al (2010) Macrophages define dermal lymphatic vessel calibre during development by regulating lymphatic endothelial cell proliferation. Development 137:3899–3910
Harvey NL, Gordon EJ (2012) Deciphering the roles of macrophages in developmental and inflammation stimulated lymphangiogenesis. Vasc Cell 4:15
Acknowledgments
The authors thank M. Kesar and staff at the Animal Facility at the Ludwig Institute for Cancer Research, Melbourne, for assistance with mouse experiments; M. Francois for provision of NG2 antibody; J. Taylor for assistance in generating figures; Y. Zhang for histology services; S. Cody for assistance with imaging; and A. Burgess and M. Hibbs for critical reading of this manuscript. Animal experiments and treatment of archived human tissue samples were in accordance with NH&MRC guidelines. This work was funded partly by a Program Grant from the National Health and Medical Research Council of Australia (NH&MRC). SAS and MGA are supported by Senior Research Fellowships from the NH&MRC. SAS would like to acknowledge the support of the Pfizer Australia Fellowship. RS is supported by the Raelene Boyle Sporting Chance Foundation and Royal Australasian College of Surgeons (RACS) Foundation Scholarship, and the RACS Surgeon Scientist Program. This work was supported by funds from the Operational Infrastructure Support Program provided by the Victorian Government, Australia.
Ethical standard
Ethics approval for research using human and animal samples was obtained from the Royal Melbourne Hospital Human Ethics Research Council, and the Ludwig Institute for Cancer Research Animal Ethics Committee, respectively.
Author information
Authors and Affiliations
Corresponding author
Additional information
SAS and MGA are consultants to Vegenics Ltd and are stock holders in Circadian Technologies.
Electronic supplementary material
Below is the link to the electronic supplementary material.
Rights and permissions
About this article
Cite this article
Shayan, R., Inder, R., Karnezis, T. et al. Tumor location and nature of lymphatic vessels are key determinants of cancer metastasis. Clin Exp Metastasis 30, 345–356 (2013). https://doi.org/10.1007/s10585-012-9541-x
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s10585-012-9541-x