Tumor Xenograft in Nude Mice

To assess the effect of Evs on the growth of CRC cells in vivo, we used 20 females athymic nude (nu/nu) BALB/c mice (aged 3–5 weeks with a weight of 22 ± 5 g) obtained from Hunan SLAC Laboratory Animal Co., Ltd. (Changsha, Hunan, China). The mice were randomly divided into four groups, five mice in each group. HCT-116 or LoVo cells adjusted to 5 × 10⁶ cells/mL were injected into nude mice (n = 5). When the xenograft tumors reached 5 mm in diameter, the nude mice were administrated with 100 μL PBS or Evs for 5 weeks (three times a week). The tumor volume was measured at an interval of 7 days according to the provided formula, tumor volume = 0.5 × a² × b, where a and b represent the short and long diameter of the tumor, respectively. The mice were euthanized 5 weeks later, and the tumor was last measured. Tumor tissues were then analyzed by immunohistochemistry. All animal experiments were performed in compliance with the Guide for the Care and Use of Laboratory Animals and carried out with the approval of the Animal Care Committee of Harbin Medical University.