Directed In-vivo lymphangiogenesis assay (DIVLA)

This assay was conducted as reported by us [29]. In brief 6 weeks old athymic nude female mice were allowed to acclimatize for 2 weeks, maintained on standard mouse chow and tap water on a 12 h light/dark cycle. Sterile angioreactors were pre-chilled at 4° C and filled with 20μl of either growth factor reduced Basement Membrane Extract (BME) (Cat# 3450-048-02, Trevigen, MD, USA) alone, or in combination with PGE2 (10μM) or EP4 agonists PGE1OH and L 902688 (10uM) or VEGF-C (20ng) (2179-VC-025, R&D Systems, CA), the latter serving as positive control for lymphangiogenesis and angiogenesis. Angioreactors were incubated at 37 °C for 1 h to allow BME gel formation, before subcutaneous implantation into the mice. Mice were anaesthetized with isoflurane. Four angioreactors containing the same agents were implanted per mouse, two on each side of the dorsal flank region, and 2 mice were used per condition. After a period of 10 days, mice were euthanized and the angioreactors were retrieved and used for three purposes as reported earlier [29]. All the angioreactors were retrieved without severing the ingrowing vessels and excising the rest of the tissues with fine scissors. One angioreactor was flash-frozen immediately with dry ice for making cryosections. The other three were used to collect cellular contents and conduct lymphatic in-growth assay and RNA extracted for real time gene expression. Three approaches were utilized for measuring angiogenesis/lymphangiogenesis: (i) Lyve-1, prox-1 and CD31 proteins by immunoflurometry of cell lysates; (ii) Lyve-1, prox-1 and CD31 mRNA by qPCR of extracted RNA; (iii) a visual quantification of lymphatic and blood vessel in-growth into the angioreactors from double labeling for Lyve-1, or Prox-1, (LEC markers) and CD31 (blood vessel endothlial marker) using the hot spot method [25, 27].