Zebrafish Model for Tumor Engraftment

Zebrafish husbandry procedures were performed according to the Directive 2010/63/EU and in compliance with local animal welfare regulations (authorization n. prot. 18311/2016; released by the “Comune di Meldola”, 9 November 2016). Ab and fli1a wild-type strain fertilized eggs were obtained and cultured according to previous works [29 (link),33 (link)]. The embryos were anesthetized with 0.02% tricaine solution before any manipulation. For DF1 engraftment, the embryos were dechorionated at 48 h postfertilization (hpf). Patient-derived tumor cells were red labelled (CellTracker™ CM-DiI, Invitrogen) using a concentration of 2.5 × 10⁵/µL. From 300 to 500 cells were injected in the yolk sack or in perivitelline space of 48 hpf embryos. DF1-grafted embryos were treated with DENO (Amgen Inc., Milan, Italy) LENVA (Eisai Ltd., Milan, Italy) and DENO + LENVA, or not treated. Embryos were exposed to the drugs at 32 °C for 72 h and imaged through a fluorescence stereomicroscope (Nikon SMZ 25 equipped with NIS Elements software) at 2, 24 and 72 hpi. Untreated embryos were also imaged using an A1 laser confocal microscope (Nikon Corporation, Tokyo, Japan), and images were analyzed with the NIS Elements software (Nikon Corporation, Tokyo, Japan).