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Quantitation of Lymphangiogenesis Using the iCycler iQ Real-Time PCR Detection System and Scorpions Detection System, Rev A

Giles Cunnick and Wen Guo Jiang, Metastasis Research Group, University Department of Surgery, University of Wales College of Medicine, Cardiff, UK


Introduction
Lymphangiogenesis refers to the formation of new lymphatic vessels. This process may occur in normal developing tissues or in tumors. In addition to spreading by direct invasion and via the bloodstream, many malignant tumors, breast cancer for example, spread via the lymphatics once new connections have been established. For this reason, the biology of tumor lymphangiogenesis has important therapeutic implications. Methods to quantitate lymphangiogenesis have not previously been described. Measuring the density of lymphatic vessels has not been reliable due to a lack of sensitive lymphatic markers and antibodies. Furthermore, it does not indicate the rate at which the production of these channels is occurring. A more accurate method would be to measure the level of mRNA for lymphatic markers in tissues, since this correlates with the rate of lymphatic synthesis.

Lymphangiogenesis has not been studied in detail, primarily due to the lack of specific markers for lymphatic endothelium. However, a novel and specific lymphatic endothelial marker has been described, called LYVE-1 (Banerji et al. 1999). This is a surface epithelial receptor located on the lymph vessel wall. The receptor appears to be highly specific for lymph vessels and is completely absent from blood vessels (Skobe et al. 2001). By quantitating LYVE-1 mRNA in tissues, estimation of the rate of lymphangiogenesis in those tissues is possible. We report a new approach to enable the quantitation of LYVE-1 mRNA in breast cancer specimens using the iCycler iQ real-
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