Detailed procedures for tissue transplantation have been previously described by Usai et al. [23 (link)]. Briefly, bulk tumor tissue screened by the histopathologist (at the Division of Surgical Pathology, University of Pisa) was washed three times with RPMI supplemented with 100 U/mL penicillin, 100 µg/mL streptomycin and 2.5 µg/mL amphotericin; then, it was minced, firstly with a scalp blade (1–3 mm), and then using the McIlwain tissue chopper (Campden Instruments LTD, Loughborough, UK) to obtain pieces of about 0.3 mm × 0.3 mm × 0.3 mm. The pieces were stained with 10 µg/mL CM-Dil (Invitrogen, Carlsbad, CA, USA) in D-PBS and incubated for 30 min at 37 °C. Tissue pieces were then washed and centrifuged three times by D-PBS and resuspended in D-PBS supplemented with 10% FBS (Gibco, Waltham, MA, USA). Pieces of fluorescent-labeled tissue were manually transplanted into the perivitelline space of n = 90 AB wild-type recipient embryos 2 days post fertilization (dpf), which were lying in 1% agarose disks in multi-well plates. After transplantation, embryos were incubated for 2 h at 35 °C. The pool of embryos xenografted with the tissue derived from each patient will be hereinafter referred to as zPDXs.
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