The nature and location of nearby lymphatics influence the metastatic propensity of a tumor
Ramin Shayan, MD1, Tara Karnezis, BSc, PhD1, Mark W. Ashton, MD2, G Ian Taylor, MD2, Marc G. Achen, BSc, PhD1, J Brian Boyd, MD3, Steven A. Stacker, BSc, PhD1.
1Ludwig Institute for Cancer Research, Melbourne, Australia, 2Brockhoff Plastic Surgery Research Unit, Royal Melbourne Hospital, University of Melbourne, Melbourne, Australia, 3Division of Plastic Surgery, David Geffen School of Medicine, University of California, LA, CA, USA.
PURPOSE: The lymphatic vasculature may be utilised by metastatic cells to transit to loco-regional lymph nodes, an important step in cancer dissemination and therefore in clinical prognostication.
The recent identified lymphatic markers have enabled the study of lymphatics in normal development and in the generation of 'neo-lymphatics' by certain tumors through secretion of protein growth factors VEGF-D and VEGF-C. Tumors exhibiting increased VEGF-D or VEGF-C expression show greater lymphatic densities and nodal spread (1).
We aimed to study lymphatics in a VEGF-D-expressing tumor model(1), xenografted to different anatomical locations in which the different lymphatic subtypes occur in different densities, in order to better understand which vessels facilitate metastasis, and the distinctions between normal and pathological lymphatics.
METHODS: We analysed lymphatics in mouse skin wounds and tissue from tumors that were generated to stably express VEGF-D or VEGF-C in the ear or flank. Using immunohistochemical lymphatic markers LYVE-1 and podoplaning we examined both the pre-existing nearby lymphatics and those generated in response to the tumor-secreted growth factors, by confocal imaging of fluorescent-labelled antibodies. We further corroberated findings in this animal model with sections of human breast and skin tissue using immunohistochemistry with the same lymphatic markers.
RESULTS: VEGF-D+ tumors that were adherent to skin induced morphologically abnormal lymphatic structures that promoted lymph node metastasis in 89% of tumors; whereas size-matched tumors that were adherent to the underlying body wall (generating similar VEGF-D levels) metastasised minimally (19%, p < 0.001) and contained no morphologically abnormal lymphatics.
Analysis of ear wounds and tumors suggested that lymphatics sprout mainly from the capillary and ‘pre-collector’ lymphatic subtypes (the small lymphatics) in skin, rather than the larger collecting lymphatics. These small lymphatics were in greater abundance (4.5-fold greater, p < 0.001) in areas closer to the surface of epithelial tissues, making tumors in these locations more likely to metastasise. This finding was replicated in the human tissues studied (4-fold, p < 0.001).
In addition, the lymphatics generated were morphologically and molecularly distinct from normal lymphatic vessel subtypes.
CONCLUSION: Whilst lymphangiogenic growth factors are a pre-requisite, anatomical tumor location dictates whether pro-metastatic neo-lymphatics are induced in response to these factors. As epithelial surfaces contain small lymphatics, tumors near a surface such as skin are likely to metastasise. This may be of prognostic significance and aid in undertanding the differences in metastasis rates between similar primary tumors. A distinct tumor lymphatic subtype may also offer new diagnostic or therapeutic molecular targets in the clinical treatment of cancer metastasis.
1. Stacker, S.A. et al., 2002.Lymphangiogenesis and cancer metastasis. Nat Rev Cancer 2:573-583.