Subcutaneous xenograft tumour model and xenograft mouse metastatic model

In order to test the ability of forming tumors in vivo, subcutaneous xenograft tumour model, which injecting stable cells into nude mice, was performed. Mice were bred and maintained under SPF conditions in the Department of Sun Yat-sen University Animal Center, as approved by the China Care Committee Institute. The female BALB/c nude mice (4–6weeks of age, 18–20 g) were randomly divided into 4 groups (n = 6/group). The stable cells (5 × 10⁶) including SiHa-NC, SiHa-TRIM62, HeLa-NC, and HeLa-TRIM62 cells were injected subcutaneously into the lower back of the female BALB/c nude mice. After injection xenograft tumours were examined twice weekly. Tumors’ length and width was measured using calipers and volumes were calculated according to the formula: length × width² × 0.52, as described previously [27]. All mice were sacrificed at the 30th day after injection. Tumors were removed from each mouse carefully, weighed and paraffin embedded. Serial 6.0 mm sections were cut and subjected to immunohistochemical staining.

Xenograft mouse metastatic model was performed for comparing the metastatic capability between different stable cell lines (SiHa-NC vs SiHa-TRIM62, HeLa-NC vs HeLa-TRIM62). The cells (2 × 10⁶/150 μL per mouse) were injected intravenously into the tail vein of female BALB/c-nu mice, respectively. After 6 weeks, the mice were sacrificed and the lungs were removed, fixed with 3.7 % formaldehyde for Hematoxylin and Eeosin (H&E) staining. The number of mice pulmonary metastatic foci was confirmed and recorded by specialized pathologists.