Bouin s solution

The Committee on the Use of Live Animals of Bengbu Medical College approved all animal experiments (LDKPZ2021-182). For the tumor metastasis assay, six-week-old female NOD/SCID mice (Hfkbio, China) were injected with 1 × 10⁶ KYSE70 cells via the tail vein (n = 6 per group). The mice were sacrificed after 6 weeks. Metastastic nodules in lung were fixed in Bouin’s solution (Applygen, China), and the number of metastases was determined. The tumor samples were embedded in paraffin, cut into 5-μm sections, and stained with hematoxylin and eosin (H&E). To evaluate tumor growth, six-week-old male BALB/c mice purchased from Hfkbio were subcutaneously injected with 1 × 10⁶ differently treated KYSE70 cells. To inhibit tumor growth, five-week-old female BALB/c mice were given a subcutaneous injection of 1 × 10⁶ KYSE70 cells. After one week, mice were equally divided into two groups according to the mean tumor volume, which was injected intraperitoneally with PBS (vehicle control) or drug regimen in PBS containing MK-8353 (10 mg/kg, three times per week, Selleck) and bevacizumab (15 mg/kg, three times per week, Genentech, USA) in PBS. Tumor size (length × width² × 0.5) was measured at the indicated time points, and the mice were sacrificed after the tumor volume reached 1.5 cm³. The weight and volume of the xenografts were measured and imaged.

Mice were purchased from and housed at Suzhou University. All animal studies were approved by the ethical regulations of Institutional Animal Care and Use Committee (IACUC) of Suzhou University. For the tumor metastasis assay, stable HCT15 clones (Control or LYAR-KD1) were injected into six-week-old female NOD/SCID mice. For each group, 10 mice were injected with 1 × 10⁶ cells per animal via the tail vein. The mice were sacrificed 6 weeks after injection, and the lung and liver metastases were examined. Metastastic nodules in lung and liver tissues were fixed in Bouin's solution (Applygen), and the number of metastases was counted. The tumor samples were embedded in paraffin, cut into 5 μm sections, and stained with hematoxylin and eosin (H&E).
For the tumor xenograft assay, 8 six-week-old female nude mice were processed by subcutaneous implantation of 1 × 10⁶ HCT15 cells expressing either control shRNA or FSCN1-shRNA. Mice were maintained for 30 days, and tumor volumes were measured at indicated time points. At the end of experiments, mice were euthanized and xenograft tumors were dissected for further analyses.