Manual microinjector

Approximately 250 KMS-28 TTA cells stably expressing DsRed fluorescent protein were injected (2 nL) into the yolk sac of each embryo with a manual microinjector (Eppendorf, Germany). Embryo were maintained at 30 °C in standard embryo medium supplemented by 0.003% PTU, 1 g L⁻¹ glucose, and 5 mmol L⁻¹l-glutamine. The efficiency of tumor xenografts was evaluated 24 h post injection (hpi) by fluorescence microscopy using ZEISS Axio Observer inverted microscope (10× magnification). Xenograft positive embryos were placed into 96-well plates (1 embryo per well) and divided randomly into four experimental groups: DMSO, 2.5 nM CFZ, 2 µM SP2509, or the combination. Tumor growth was evaluated 72 hpi by fluorescence microscopy. Tumor xenograft volume of control and drug-treated animals was estimated at 72 hpi by measuring the area of fluorescence on photomicrographs and normalized to the signal obtained at 24 hpi. Images were processed using ImageJ program. All procedures were authorized by the Ethical Committee of the University of Torino and the Italian Ministry of Health.

Zebrafish embryos at 2 dpf were manually dechorionated and anaesthetized with 0.016% tricaine (Ethyl 3-aminobenzoate methanesulfonate salt; Sigma-Aldrich). EVs derived from the Caco-2 and SK-CO-1 lines were resuspended in PBS 1X and injected into the duct of Cuvier, to allow their delivery in the circulation. A manual microinjector (Eppendorf, Germany) was used with glass microinjection needles. Control embryos were injected with serum-free media (DMEM for Caco-2 and EMEM for SK-CO1 cells). Following the injections, embryos were kept at 28 °C for 20 h, until their processing for RNA extraction.