Nsg mice

NSG mice (3–4 weeks old, female, 15–20 g) used in this study were obtained from Shanghai Model Organisms Center. A total of 5 × 10⁶ RPMI8226‐Luciferase cells in 200 μL medium were mixed with isopycnic matrigel (Corning Incorporated) and implanted subcutaneously into the abdominal wall of mice. Ten days after tumor implantation, treatment groups of mice (n = 6 per group) received tail intravenous injection of CAR‐T or control T cells at Days 12 and 17 with a total of 5 × 10⁶ T cells per mice (about 2 × 10⁶ transduced cells). Mice in blank group were untreated. Animal experiments were conducted in accordance with the Declaration of Helsinki and approved by the Institutional Research Ethics Committee of the First Affiliated Hospital of Zhengzhou University (Lot No. 2022‐KY‐1062). Mice were given D‐Luciferin (150 mg/kg, i.p.) and anesthetized with isoflurane. After 5 min, luminescence was detected using the in vivo imaging system (IVIS) and the intensity was quantitated and normalized by the Living Image software (PerkinElmer).

786-O and Caki-1 subcutaneous tumor-bearing male NSG mice were purchased from the Shanghai Model Organisms Center and randomly assigned to groups based on tumor size. In the siRNA xenograft tumor model group (n = 6, each group), cholesterol-modified circPDHK1-siRNA (10 nmol, GenePharma) was intratumorally injected once every two days for five consecutive weeks. Tumor volumes were measured every 2 d. Tumor tissues were excised and weighed after mice were euthanized, fixed, and stained for immunohistochemistry. In the xenograft tumor model overexpression group (n = 5, each group), a lentiviral vector containing cloned inserts (Oligobio, Beijing) was injected intratumorally for 18 days. Tumor volume measurements and processing were carried out as described above. For the lung metastasis model, stably transfected Caki-1 cells (6 × 10⁶ cells/0.2 mL PBS) were injected into the tail vein of 4-week-old male mice. After 48 days, the mice were assessed using the Aura Spectral Instruments in vivo Imaging System and sacrificed. Lung tissues were excised, photographed, and stained with hematoxylin and eosin (H&E), and the lung metastatic nodes were observed. All the animal experiments were conducted according to the protocol approved by the Ethics Committee of Chongqing Medical University (approval number: 2022013).

NSG mice (6–8 weeks old) were purchased from SHANGHAI MODEL ORGANISMS (Shanghai, China). All animal experiments were approved by the Animal Care and Use Committee at Xuzhou Medical University. The stable cells were resuspended in a serum-free medium (5 × 10⁷ cells /mL). NSG mice were randomly divided into the control group (Vector), TRIM21 overexpression group (OE-TRIM21), SREBF1 overexpression group (OE-SREBF1), and TRIM21 + SREBF1 double overexpression group (OE-TRIM21 + OE-SREBF1). Mice were anesthetized with isoflurane, after the kidney was exposed from the posterior side, the cell suspension (5 × 10⁵ cells /10μL) was injected under the renal capsule, and the wound was sutured and sterilized. Every 7 days in vivo imaging of small animals was used to monitor tumor size and metastasis. The experiment was terminated when the difference in tumor growth between groups was fully developed. The orthotopic tumors were collected for frozen sections to detect the formation of lipid droplets. Paraffin embedding and histochemical staining were performed to detect the expression of lipid metabolism-related indicators.

All procedures performed on animals were in accordance with regulations and established guidelines and were reviewed and approved by the Institutional Animal Care and Use Committee at the Shanghai Institute of Materia Medica, Chinese Academy of Sciences (IACUC Issue NO. 2022-01-JHL-27 for NSG mice; IACUC Issue NO. 2021-03-JHL-22 for BALB/c mice). NSG mice were obtained from Shanghai Model Organisms Center, Inc; BALB/c mice were obtained from Beijing Huafukang Biotechnology Co. Ltd (Beijing, China). Six- to eight-week-old mice were used for the studies and were maintained with free access to pellet food and water in plastic cages at 21 ± 2 °C and humidity (50 ± 10%) conditions and kept on a 12 h light/dark cycle. The tumor size tolerated by the xenograft tumor model mice did not exceed 2000 mm³, the maximal tumor burden permitted by the Institutional Animal Care and Use Committee at the Shanghai Institute of Materia Medica, Chinese Academy of Sciences.

All animal experiments were performed according to the protocols approved by the Institutional Animal Care and Use Committee (IACUC) of Southern Medical University. For xenograft tumor growth, 5 × 10⁶ cells were injected subcutaneously into the left (control group) and the right (SLC35B4 knockdown group) flanks of the immunodeficient NSG mice (purchased from Shanghai Model Organisms, Shanghai, China. n = 6), respectively. For inducible gene knockdown, the drinking water containing 2 mg/L of doxycycline was supplied with doxycycline (20 mg/kg body weight) injected intraperitoneally every day.
After obtaining adequate informed consent, HCC tissue and adjacent normal tissue (ANT, exceeding the edge of the tumor by at least 2 cm) were obtained from HCC patients who underwent curative resection for HCCs in Nanfang Hospital of Southern Medical University, Guangzhou, China, between November 2010 and May 2015. This study was approved by IRB of Nanfang Hospital at Southern Medical University and was performed according to the Declaration of Helsinki (6th revision, 2008).