Xenograft mouse models

Ten 6-week-old female NOD/SCID mice weighing approximately 20 g were purchased from Hunan SJA Laboratory Animal Co., Ltd. and were randomly divided into two groups, five in each group. The mice were kept in SPF animal rooms with five mice in each cage in Nanchang Royo Biotech Co., Ltd. The constant temperature of the environment was 22 °C to 25 °C, and the humidity was approximately 40%. Additionally, the mice were fed with appropriate irradiated feed and sterile water.

5-FU has been widely used as a conventional chemotherapeutic agent for the clinical treatment of gastric cancer (Mahlberg et al., 2017). Therefore, we selected 5-FU, a representative chemotherapeutic drug, to further verify whether the drug resistance of gastric cancer cell lines with high ARK5 expression was higher than that of parental cell lines in vivo. First, during the experiment, SGC7901 cells and SGC7901/DDP cells were digested with trypsin and then were resuspended at 2 ×10⁷ cells/ml with culture medium. Additionally, the mice were anesthetized with 1% pentobarbital sodium (0.1 ml/20 g) and each was inoculated subcutaneously with 2 ×10⁶ cells in 100 µl of medium. On the 10th day postinjection, small lumps of the same size could be observed on each mouse, confirming successful inoculation. Thereafter, mice in both the SGC7901 and SGC7901/DDP groups were intraperitoneally injected with anesthetic pentobarbital sodium and 20 mg/kg of 5-FU twice a week for three weeks. Particularly, the weight and tumor size of mice were measured with a sterile Vernier caliper every three days, and the volumes of tumors were calculated using the following formula: Volume = (length ×widt h²)/2 (Von Kalle et al., 1986), where the length and width refer to the maximum and minimum diameters, respectively. On the 31st day postinjection, the mice were weighed for the last time, and then were anesthetized with pentobarbital sodium and sacrificed by breaking the neck. Finally, the tumors were separated from the subcutaneous region of the mice with sterile surgical instruments, and the weight and volume were measured.

All animal procedures were approved by the Laboratory Animal Ethics Committee of Nanchang Royo Biotech Co., Ltd. (Nanchang, China; approval number: RYE2018081801), and all laboratory mice were treated strictly according to the Institutional Animal Care’s guidelines in the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996).