Models and Technologies An In Vivo Antilymphatic Screen in Zebrafish Identifies Novel Inhibitors of Mammalian Lymphangiogenesis and Lymphatic-Mediated Metastasis Jonathan W. Astin 1 , Stephen M.F. Jamieson 2,3 , Tiffany C.Y. Eng 1,2 , Maria V. Flores 1 , June P. Misa 1 , Annie Chien 1 , Kathryn E. Crosier 1,3 , and Philip S. Crosier 1,3 Abstract The growth of new lymphatic vessels (lymphangiogenesis) in tumors is an integral step in the metastatic spread of tumor cells, first to the sentinel lymph nodes that surround the tumor and then elsewhere in the body. Currently, no selective agents designed to prevent lymphatic vessel growth have been approved for clinical use, and there is an important potential clinical niche for antilymphangiogenic agents. Using a zebrafish phenotype-based chemical screen, we have identified drug compounds, previously approved for human use, that have antilymphatic activity. These include kaempferol, a natural product found in plants; leflunomide, an inhibitor of pyrimidine biosynthesis; and cinnarizine and flunarizine, members of the type IV class of calcium channel antagonists. Antilymphatic activity was confirmed in a murine in vivo lymphangiogenesis Matrigel plug assay, in which kaempferol, leflunomide, and flunarizine prevented lymphatic growth. We show that kaempferol is a novel inhibitor of VEGFR2/3 kinase activity and is able to reduce the density of tumor- associated lymphatic vessels as well as the incidence of lymph node metastases in a metastatic breast cancer xenograft model. However, in this model, kaempferol administration was also associated with tumor deposits in the pancreas and diaphragm, and flunarizine was found to be tumorigenic. Although this screen revealed that zebrafish is a viable platform for the identification and development of mammalian antilymphatic compounds, it also highlights the need for focused secondary screens to ensure appropriate efficacy of hits in a tumor context. Mol Cancer Ther; 13(10); 2450–62. Ó2014 AACR. Introduction Cancer is now the leading cause of death worldwide. The majority of cancer-induced deaths are caused by metastatic malignancy, a process by which cancer cells spread through the body via the lymphatic or blood vasculature. The importance of lymphatic metastases is well recognized in cancer staging and treatment, with the spread of cancer cells to surrounding lymph nodes being associated with poor prognosis in many cancers (1, 2). It has been estimated that more than 80% of solid tumors metastasize, at least partially, through the lym- phatic vasculature (3). Tumor cells can enter the lymphat- ic vasculature by either invading preexisting lymphatic vessels present in the tissue surrounding the tumor, or by promoting lymphangiogenesis and creating new lym- phatic vessels within and around the tumor—a process termed tumor-induced lymphangiogenesis (4, 5). The increase in lymphatic vessel density in the tumor has been proposed to facilitate the metastatic spread of cancer cells, as it has been correlated with an increased incidence of lymph node metastases and a consequential decrease in patient survival in many cancers (3, 6). As well as being implicated in cancer metastasis, de novo lymphangiogen- esis is also associated with host rejection of renal trans- plants (7) and corneal grafts (8). Studies have shown that the receptor tyrosine kinases VEGFR2 and VEGFR3 as well as their ligands VEGF-A, VEGF-C, and VEGF-D play important roles in tumor- induced lymphangiogenesis (4, 9–12). It is known that inhibition of the VEGFR signaling pathway reduces tumor-associated lymphangiogenesis and lymph node metastasis in animal models (13–16) and also improves the survival of corneal transplants (8). Therefore, there is a need to develop and test not only inhibitors of VEGFR/ VEGF signaling but also to identify novel antilymphatic drugs that act outside of this pathway. Zebrafish are a powerful model organism in which to study lymphangiogenesis. Lymphatic vessels can be 1 Department of Molecular Medicine and Pathology, School of Medical Sciences, University of Auckland, Auckland, New Zealand. 2 Auckland Cancer Society Research Centre, School of Medical Sciences, University of Auckland, Auckland, New Zealand. 3 Maurice Wilkins Centre for Molec- ular Biodiscovery, School of Biological Sciences, University of Auckland, Auckland, New Zealand. Note: Supplementary data for this article are available at Molecular Cancer Therapeutics Online (http://mct.aacrjournals.org/). Corresponding Author: Philip S. Crosier, School of Medical Sciences, University of Auckland, Private Bag 92019, Auckland 1142, New Zealand. Phone: 64-9-923-6279; Fax: 64-9-373-7010; E-mail: ps.crosier@auckland.ac.nz doi: 10.1158/1535-7163.MCT-14-0469-T Ó2014 American Association for Cancer Research. Molecular Cancer Therapeutics Mol Cancer Ther; 13(10) October 2014 2450 Downloaded from http://aacrjournals.org/mct/article-pdf/13/10/2450/2326851/2450.pdf by guest on 10 June 2022