Tumorigenicity assay in vivo

Cancer stem cells (1 × 10⁴ cells/mL) or cancer non-stem cells (1 × 10⁴ cells/mL) were mixed with Corning Matrigel matrix (Corning, USA) at a ratio of 1:1. Subsequently, 100 μL of cell suspension was subcutaneously injected into BALB/C nude mice (4 weeks old) to induce tumor growth. The tumor volume was measured once every 3 days. Forty days later, the mice were sacrificed to determine the size and weight of solid tumors.

The DOCK2 edited cells (10⁶ cells/mL) and the DOCK2 unedited cells (10⁶ cells/mL) were mixed with Corning Matrigel matrix (Corning) at a ratio of 1:1, respectively. Subsequently, 100 μL of cell suspension was subcutaneously injected into BALB/C nude mice (4 weeks old) to induce tumor growth. The tumor volume was measured once every 3 days. Forty-five days later, the nude mice were sacrificed. The sizes and weights of solid tumors were determined.

To evaluate the role of miR-17 in tumorigenesis in vivo, MDA-MB-435 stem cells (10⁶ cells/mL) were subcutaneously injected into BALB/C nude mice. Three days later, the mice were subcutaneously and intravenously injected with miR-17, miR-17-scrambled, AMO-miR-17, or AMO-miR-17-scrambled at 80 mg/kg once every 3 days. The tumor volume was examined every 3 days. Thirty days later the mice were sacrificed, followed by examination of tumor sizes and weights of solid tumors.

Animal experiments were approved by The Animal Experiment Center of Zhejiang University, China. All methods were conducted in accordance with the approved guidelines.