Four- to eight-week-old female BALB/c nude mice were purchased from the Model Animal Research Center of Nanjing University (Nanjing, China). For the tumor-limiting dilution assay, 1 x 10⁷, 5 x 10⁶, and 2.5 x 10⁶ of LV3-miR-375 cells, LV3-SLC7A11 cells, LV3-shSLC7A11 cells, LV3-miR-375-SLC7A11 cells, and LV3-NC cells (BGC-823 and SGC-7901) were subcutaneously implanted into the underarm of mice. Fifteen days later, all mice were euthanized and tumor tissues were collected and weighed. For metastasis experiment, mouse models were established by intravenous injection of cells. Three mice per group were injected with 2 x 10⁶ cells in 200 uL RPMI-1640 serum-free media. Six weeks after injection, mice were sacrificed for collecting lung tissues.

The 4-week-old female BALB/c nude mice were used for constructing xenograft model. HeLa cells (1 x 10⁷ cells/mL) were injected into the dorsal flanksusing using 1-mL syringes. Then tumor size was measured and mice received intertumoral injection of si-crEPSTI1-1 (40 uL siRNA1 for cicrEPSTI1) and negative control (40 uL negative control) every four days, respectively (5 mice/group). After 28 days, the mice were sacrificed and xenografts were measured.