Following the fifth sorting cycle, A375 cells were first cultivated to dispose the sorting particles, harvested, and injected (5 × 106 cells in 100 μl of PBS containing 5% Matrigel) subcutaneously to male athymic nude–Foxn1 HSD 5- to 6-week-old mice, under isoflurane anesthesia. Mice were inspected and weighed every other day. From day 7 of the injection, tumors were measureable and their volume calculated through the ellipsoid equation (square of the tumor width and multiplied by 0.52). Twenty-two days after the injection, mice were euthanized by cervical dislocation under ketamine/xylazine sedation, and tumors were extracted, weighed, and photographed. Tumors were fixated in formalin and embedded in paraffin for histological examinations.

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