Tissue samples (primary tumor histologically confirmed to be not significantly contaminated by normal tissues, necrotic tissues and lymphocytes, and histologically normal tissue adjacent to the tumor), were rapidly frozen in liquid nitrogen. Within 30 min from surgery, a tumor tissue portion, was placed in Advanced DMEM/F12 medium with 5% fetal bovine serum (FBS) (Gibco), 2 mM glutamine and an antibiotic/antifungal solution (Gibco) containing 10,000 units/mL of penicillin, 10,000 µg/mL of streptomycin, and 25 µg/mL of amphotericin B, and immediately transferred to the laboratory for subsequent processing. Tumor tissue was manually separated from adipose areas using sterile scalpel and was minced into pieces < 1 mm3 with sterile scissors; the pieces were placed in a sterile microblade-equipped polyethylene chamber, medicon (Becton–Dickinson) with medium mentioned above. When the medicons were inserted into the Medimachine (Becton–Dickinson), the fragments came into contact with the blades and were dissociated from four to five times for 20 s at a constant speed < 100 r.p.m12. Medium containing recovered cells was filtered by porous polyester membranes (Becton–Dickinson). After filtration, cells were washed and transferred into a 1 mL plate and observed under an inverted microscope (Leitzs, Germany) in order to check the presence of organoids; subsequently organoids were separated from the other cellular fractions by centrifugation at 700 rpm for 2 min. Pellet, suspended in complete culture medium, was incubated at 37 °C in a humidified atmosphere at 5% CO2. After 24 h, organoids and single cells started the attachment process, and the supernatant, loaded in organoids and non-adherent cells, was transferred on a new plate. This operation was repeated every 48–72 h to get a cellular culture ever cleaner and to increase the number of available cellular samples. Differential trypsinization was used to get rid of the fibroblasts13. Briefly, cells were incubated with trypsin for a 1 min, until detachment of fibroblasts. Then, fibroblasts were discarded and the dish was washed 2–3 times with Advanced DMEM/F12 medium before adding fresh complete medium to the cells. Cultures were observed daily for cell growth and the medium was changed every three days (Supplementary Fig. S1).

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