E3 medium

Wild-type Tubingen (TU) and Wild Indian Karyotype (WIK) Danio rerio adult zebrafish lines were maintained and bred according with standard protocols. Their embryos were raised under a 14-h light, 10-h dark cycle in 28 °C E3 medium (in mM: 5 NaCl, 0.17 KCl, 0.33 CaCl₂, and 0.33 MgSO₄; Sigma-Aldrich Corp., St. Louis, MO, USA) and staged according to developmental stage in days post fertilization (dpf). At 3–4 dpf they were transferred to 24-wells plates with E3 solution and maintained there until the experiment occurring the following day.

For both human and mouse tissue samples, cryomicrotome-cut transversal sections (10 μm) were collected on polylysine slides and stored at −80° C before staining. Briefly, mouse and human tissue sections were blocked with 5% normal goat serum containing 0.1% Triton X-100 for 60 min (room temperature). Sections were incubated overnight with the following primary antibodies: collagen IV (Southern Biotech, Birmingham, AL, USA); ADAMTS-5, MOMA-2, versican-neo (the DPEAAE neoepitope of versican, neoepitope on versican generated by ADAMTS cleavage), and aggrecan (Abcam, Cambridge, MA, USA); aggrecan-neo (the NITEGE neoepitope of aggrecan) and versican (Thermo Fisher Scientific, Waltham, MA, USA). The sections were then incubated with the appropriate secondary antibody (Alexa Fluor 488, 594, or 647, and FITC or Cy3, Abcam). The sections were washed and coverslipped with antifade medium containing DAPI (Abcam), and images were digitally captured using an Olympus FluoView 1200 confocal microscope, followed by analysis using Image J (NIH, USA) software.
For zebrafish, living larvae were embedded in 1.2% low melting agarose, which was dissolved in E3 medium and Tricaine (Sigma-Aldrich). Tg (flk: EGFP; gata1: DsRed) embryos at 48 hpf with intracranial hemorrhage were captured by confocal microscopy (Carl Zeiss, Jena, Germany).

First zebra fish embryos were injected at 48 h post fertilization with approximately 500 stained OCI-LY3 tumor cells in 5 nL medium in the egg yolk to validate if the cell line could form tumors. The tumors were photographed and embryos transferred to individual wells in a 24-well plate in 500 µL of PTU-treated, E3 medium (Sigma-Aldrich). Tumors were visualized again at 24 and 72 h post implantation by fluorescence microscopy. Tumor cells were analyzed with a software designed to assess tumor size in zebra fish embryos (Bioreperia, Linköping, Sweden). Tumor growth was determined by normalizing the area of the tumors at each given time point to that of the same tumor at day 0. Tumor cells grew well in the yolk and the volume increased by 70% between day 0 and day 3.
In the next step, zebra fish embryos (n = 4 × 21) were injected with tumor cells as above. KAN0441571C was added to the medium at 4 different concentrations: 25, 100, 250, 1000 nM. One group (n = 21) was untreated. Tumors were visualized by fluorescence microscopy (photographed) at day 0 and at 24 and 72 h post implantation. Tumor size was determined as above.

Treated embryos were examined for survival rates at 24, 48, 72, and 96 hpf using an Olympus stereoscope (Olympus, Tokyo, Japan). Each embryo was anesthetized with 0.02% tricaine (Sigma-Aldrich Corp., St. Louis, MO, USA) in an E3 medium, mounted with 3% methylcellulose (Sigma-Aldrich Corp., St. Louis, MO, USA). The bright field images were captured using an Olympus stereoscope (Olympus, Tokyo, Japan) with an AxioCam GRC camera (Carl Zeiss, Overkochen, Germany) using the Zeiss Zen 3.4 (Blue edition) software [27 (link)]. The body length and brain size of the embryos were measured using polygon selection in ImageJ software (http://rsbweb.nih.gov/ij/, accessed on 28 December 2021). All experiments were repeated at least thrice.

Wild-type adult WIK danio rerio zebrafish lines were fed and maintained in line with standard protocols. Their embryos were incubated and raised under a 14-10-hours light-dark cycle in 28°C in 1.7L transparent breeding tanks. At 3–4 days past fertilization, they were transferred to 35x12 mm cell culture plates with E3 medium (solution in mM: 5 NaCl, 0.17 KCl, 0.33 CaCl₂, and 0.33 MgSO₄; Sigma-Aldrich Corp., St. Louis, MO, USA) and maintained there until the start of the experiment the following day.