After the experiment, tumor tissues were removed, and the tissues were cut into 5 mm-thick strips and placed into a digestive solution containing 2 mg/mL collagenase IV (VICMED, Xuzhou, China) and 0.25 mg/mL hyaluronidase (MCE) in RPMI-1640 culture medium (KeyGEN Biotechnology Co, Nanjing, China) 37 . The tissues were incubated at 37 °C with continuous shaking at 180 rpm for 60-120 min and a gentle vortex every 15 min. The resulting cell suspensions were strained through a 70 μm cell strainer, pelleted, washed twice in PBS and sorted by flow cytometry 38 (link).
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