2.13. Xenograft tumours and bioluminescent imaging

Animal studies were performed according to the protocols approved by the Institutional Animal Care and Use Committee of Guangdong Provincial Hospital of Chinese Medicine (Ethics Approval Number 2017037) and the National Institutes of Health guidelines for the care and use of Laboratory animals (NIH Publications No. 8023, revised 1978). Nude mice (aged 4‐6 weeks) purchased from Beijing Vital River Experimental Animal Co. Ltd. were maintained at the Animal Center of Guangdong Provincial Hospital of Chinese Medicine. A549‐Luc or A549PDPK1+/+‐Luc (2 × 10⁶) were injected subcutaneously into the nude mice. Xenografts were allowed to grow when the initial measurements were made with calipers. For bioluminescence imaging (BLI), mice were anesthetized by inhalation of 2% isoflurane. The substrate D‐luciferin (Caliper Life Sciences) was injected into the peritoneal cavity of the mice at a dose of 150 mg/kg. The intensity of the BLI signal was determined using the IVIS‐200 Imaging System (Xenogen/Caliper). Mice were then randomly divided into the control and SM groups (n = 10/group), and SM was injected intraperitoneally daily at a dose of 8 mg/kg for up to 25 days based on our previous reports and others,7, 34, 35 which showed significant inhibitory effect of SM on tumour growth without apparent toxicity. Tumour volume was calculated using the formula for a spheroid: volume = (width² × length) × 0.5. Bioluminescence is expressed as photons/s. Body weights of mice were measured once a week. All mice were sacrificed on the 25th day in accordance with the Guidelines for the Care and Use of Laboratory Animals. At the end of the experiments, xenograft tumours were isolated, and expression of HOTAIR, miR‐214‐3p and PDPK1 was determined by qRT‐PCR as well as Western blot.