Zebrafish xenograft and metastasis assay

A detailed description of the experimental procedure for this assay is provided at Bio-protocol (Paatero et al., 2018). Adult zebrafish (Danio Rerio) of casper strain (roy-/-; mitfa-/-) (White et al., 2008) were maintained according to standard procedures (Nuesslein-Volhard and Dahm, 2011; Westerfield and Zon, 2009) and embryos were collected after natural spawning. Experimentation with zebrafish was performed under licence ESAVI/9339/04.10.07/2016. The zebrafish embryos were cultured in E3-medium (5 mM NaCl, 0.17 mM KCl, 0.33 mM CaCl₂, 0.33 mM MgSO₄) supplemented with 0.2 mM phenylthiourea (PTU, Sigma-Aldrich) at 33°C. Two days post-fertilization, the embryos were anesthesized with MS-222 (200 mg/l, Sigma-Aldrich) and mounted into low-melting point agarose for tumor transplantation. Prior to transplantation, the co-cultured and siRNA-treated WM852-GFP melanoma cells were prepared and separated from LECs as described above. Approximately 5–10 nl of melanoma cell suspension was microinjected into pericardial cavity of the embryo using CellTramVario (Eppendorf), Injectman Ni2 (Eppendorf) micromanipulator and borosilicate glass needles pulled from glass capillaries (TW100-4, World Precision Instruments Ltd., Sarasota, FL) using micropipette puller (PB-7, Narishige, Tokyo, Japan). After transplantation, the embryos were released from the agarose and cultured in E3-PTU at 33°C. On the following day, the successfully xenografed healthy embryos were selected to the experiment and placed into 12-well plates (1 embryo per well). At 6 dpf (4 days post-injection) the embryos were anaesthesized with MS-222 and imaged in lateral orientation with Zeiss StereoLumar V12 fluorescence microscope.

The circularity and area of the primary tumor was measured manually using FIJI software (ImageJ version 1.49 k) (Schindelin et al., 2012). In cases where embryo carried more than one primary tumor, the largest nodule was considered as primary tumor and measured, or in cases where equally sized nodules existed, all of them were measured. The number of invaded cells were counted manually based on GFP-fluorescence. Only invading cells outside the pericardial cavity were counted. Invading cells above the yolk sac or in the lens were not also counted as these sites tend to have prominent autofluorescence. Samples having significant malformations and images where embryo was not laterally oriented were excluded from the analysis. Samples were not blinded for imaging and subsequent analyses.