Balb c nude mice

Forty BALB/c nude mice (five-week old, 15± 1 g) were purchased from the Model Animal Research Center of Nanjing University (Nanjing, China). All animal experiments (Approval No. IACUC-20201231-04) were conducted according to the relevant laws and institutional guidelines with the approval of the Animal Ethics Committee of Jinan University [SYXK (YUE) 2017-0174]. After acclimation for 10 days, the mice were randomly divided into two groups and inoculated (sc) with HT29 or HCT116 cells (1×10
⁷ cells/ 100 μL each) in the right forelimb pit. When the tumor grew to 50 mm
³, the mice in each group were randomly assigned into the following two subgroups (
n=10): the control group (solvent) and the GRh3-treated group (ig, 20 mg/kg/d). The body weights and tumor volumes were measured every 7 days, and the tumor size was calculated using the following equation: tumor volume=length×width
²×1/2. Twenty-one days after the treatment, the mice were anaesthetized with pentobarbital (ip, 150 mg/kg) and sacrificed by cervical dislocation. The serum was retained, and the xenograft tumor tissues, liver and kidney were resected for subsequent experiments.

Four weeks old female BALB/c nude mice were purchased from Model Animal Research Center of Nanjing University and housed in a pathogen-free environment in the Animal Laboratory Unit of Nanjing University. For the xenograft model, GPER1 knockdown and control GC cells (2 × 10⁶) resuspended in 200 μL of PBS were injected in the flank of nude mice (5 mice/group). These mice were sacrificed 21 days after tumor cell implantation. At autopsy the tumor was removed and weighed. For peritoneal dissemination model, GPER1 knockdown and control GC cells (3 × 10⁶) in 400 μL PBS were injected into the peritoneal cavity. Peritoneum metastasis was examined and recorded when mice were killed at 14 days after injection.

Four-week-old female BALB/c nude mice were purchased from The Model Animal Research Center of Nanjing University, Nanjing, China. For in vivo chemosensitivity analyses, mice were randomly separated to four groups (9 mice per group). HepG2-EGFP, HepG2-VASH2, HepG2-shcont, and HepG2-shVASH2 cells were subcutaneously injected into the flanks of the mice (10⁶ cells/100 µl per flank). After injection with liver cancer cells for 9 days when the tumor volume has not statistically significant, each group of the mice were further divided into two subgroups, CDDP(−) and CDDP(+). The mice in CDDP(+) group began receiving CDDP (10 mg/kg) (Nanjing, China) intraperitoneally every 3 days [29] (link),while CDDP(−) group was not received this treatment. Tumor volume was calculated using the formula (W²×L)/2, where L is the length of the tumor and W is the width of the tumor. Bidimensional tumor measurements were conducted with calipers every 3 days. After six 3-day cycles of treatment, all nude mice were sacrificed and tumors were excised for further study.

A total of 10 BALB/c nude mice were purchased from Model Animal Research Center of Nanjing University (Nanjing, China). 2 × 10⁶ stably circ‐TSPAN4 knockdown H1993 cells and control cells were tail vein injected into nude mice, respectively. Lung metastasis was monitored by the IVIS Lumina II system. After 6 weeks, all mice were sacrificed and the lung tissues were collected for Haematoxylin‐eosin staining. All protocols were approved by the Institutional Animal Care and Use Committee review board of Hangzhou Cancer Hospital.

For the xenograft tumor model, 4-week-old male BALB/c nude mice were purchased from the Model Animal Research Center of Nanjing University (Nanjing, China) and raised under specific pathogen-free (SPF) conditions according to the National Institutes of Health (NIH) guidelines for the care and use of mice. Mice were randomly divided into 2 groups (n = 5 for each group) and subcutaneously injected with A549 cells (5 × 10⁶ cells per mouse) that were transfected with LINC02159 siRNA or control vector. The tumor size was measured every 3 days, and the formula for calculating tumor volume was: volume = 0.5×length×width². Tumor tissues were harvested for hematoxylin and eosin (H&E) and immunohistochemistry (IHC) staining. The animal study has been approved by Jiangsu University Animal Use and Care Committee.