Cell experiment:

The transgenic lines Tg(fli1:GFP) and Tg(mpx:GFP) are used in this study. 0.2 mM N-phenylthiourea (PTU) is applied to prevent pigment formation from 1 day post-fertilization (dpf). For Pu.1 knockdown, Pu.1 MO (1 mM for partial knockdown and 2 mM for complete knockdown is injected into the yolk at the one-cell stage. For pharmacological inhibition, the VEGFR tyrosine kinase inhibitors KRN633 (0.1-1 μM) or Sunitinib (0.1-1 μM), Beclomethasone (25 μM) and β-amino-proprionitrile (βAPN, 500 μM) are applied directly to the egg water and refreshed every 2 days. For pharmacological inhibition, Beclomethasone is applied to the embryos 4 h before implantation and KRN633, Sunitinib and βAPN are applied 4-6 h post-implantation. For each cell line or condition, data are representative of ≥ three independent experiments, with ≥30 embryos/group. Experiments are discarded when the survival rate of the control group is<90%[1].

Beclometasone (Beclomethasone) is a prototype glucocorticoid receptor agonist.

An inhibition of the normal physiological neutrophil migration and of neutrophil chemotaxis directed by wounding-induced inflammation is detected at 4 h after administration of 25 μM Beclomethasone. Tumour cell invasion and micrometastasis is also reduced in embryos incubated in 25 μM Beclomethasone 4 h before implantation. In addition, the lysyl oxidase inhibitor β-aminoproprionitrile (βAPN) largely reduces fibrillar collagen and enhances the CHT-TF transmigration of neutrophils, leading to a significant increase of tumour cell invasion and subsequent formation of micrometastases. Notably, βAPN inhibits neutrophil chemotaxis induced by inflammation, indicating that the increase of tumour cell invasion in βAPN-treated embryos is correlated with enhanced non-pathological neutrophil migration, but not with inflammation[1].

[1]. He S, et al. Neutrophil-mediated experimental metastasis is enhanced by VEGFR inhibition in a zebrafish xenograft model. J Pathol. 2012 Aug;227(4):431-45.