Mating was setup 2 days prior to injection day and zebrafish embryos were collected and incubated in E3 embryo medium (5 mM NaCl, 0.17 mM KCl, 10 mM HEPES, 0.33 mM MgSO4·7H2O, 0.33 mM CaCl2·6H2O, and 0.00001% methylene blue) containing 0.2 mM N-phenyl-thiourea (PTU) (catalog no: P7629, Sigma-Aldrich). Two days post-fertilization, the chorion was removed manually using fine forceps, and the embryos were anesthetized using E3 medium containing 200 mg/L Ethyl 3-aminobenzoate methanesulfonate (Tricaine) (catalog no: A5040, Sigma-Aldrich). Anesthetized embryos were mounted in 0.7% low melting agarose containing 200 ug/ml of Tricaine and were microinjected with 500 cells in the yolk sac using Nanoject III (catalog no: 3-000-207; Drummond Scientific Company, PA, USA). At 1 day post-injection (dpi), embryos with similar graft size were selected and imaged using both bright field and RFP channels and incubated in E3-PTU medium containing 5 ug/ml doxycycline at 34 until further imaging. At 4 dpi, embryos were anesthetized and imaged again using both bright field and RFP channels using Olympus IX-73 microscope. Cells that migrated outside the yolk sac (injection site) were represented by a notable fluorescent dot and considered a metastatic event; these were counted manually for all embryos in each experimental group.

Mating was setup 2 days prior to injection day and zebrafish embryos were collected and incubated in E3 embryo medium (5 mM NaCl, 0.17 mM KCl, 10 mM HEPES, 0.33 mM MgSO4·7H2O, 0.33 mM CaCl2·6H2O, and 0.00001% methylene blue) containing 0.2 mM N-phenyl-thiourea (PTU) (catalog no: P7629, Sigma-Aldrich). Two days post-fertilization, the chorion was removed manually using fine forceps, and the embryos were anesthetized using E3 medium containing 200 mg/L Ethyl 3-aminobenzoate methanesulfonate (Tricaine) (catalog no: A5040, Sigma-Aldrich). Anesthetized embryos were mounted in 0.7% low melting agarose containing 200 ug/ml of Tricaine and were microinjected with 500 cells in the yolk sac using Nanoject III (catalog no: 3-000-207; Drummond Scientific Company, PA, USA). At 1 day post-injection (dpi), embryos with similar graft size were selected and imaged using both bright field and RFP channels and incubated in E3-PTU medium containing 5 ug/ml doxycycline at 34 until further imaging. At 4 dpi, embryos were anesthetized and imaged again using both bright field and RFP channels using Olympus IX-73 microscope. Cells that migrated outside the yolk sac (injection site) were represented by a notable fluorescent dot and considered a metastatic event; these were counted manually for all embryos in each experimental group.