Cell purification and culture

Murine CD8+ T cells were isolated from spleen by using a positive selection kit (STEMCELL). Cells were stimulated and cultured with plate-bound anti-CD3 (312.5 ng/cm2) and soluble anti-CD28 (0.5 µg/mL) antibodies. At day 3 and day 5, cells were transferred to new wells and cultured in standard T-cell medium with 30 U/mL mouse IL-2 for another 2 days.

For the treatment, DMSO dissolved AA, GA, sulfo-N-succinimidyloleate (SSO), and ferroptosis inhibitors (DFO) were added into medium on day 6 and cultured for another 24 h.

U937 cells (ATCC, Manassas, VA, USA) were grown in RPMI1640 Medium (HyClone Laboratories, Logan, UT, USA) with 10% fetal bovine serum (FBS) (Biological Industries, Beit Haemek, Israel) with or without the indicated treatments.

Small interfering RNA (siRNA) transfection

Cell lines were transfected with 6 nM small interfering RNA (siRNA) targeting CD36 expression (Santa Cat. #sc-29995) using Lipofectamine® RNAiMAX transfection reagent (Invitrogen Cat. #13778030) according to the manufacturer’s protocol.

Flow cytometry

Experiment was performed according to the manufacturer’s protocol [14]. Briefly, for lipid peroxidation and cellular iron measurement, cells were washed with phosphate buffered saline (PBS) and then incubated in a humidified chamber at 37 ℃ with 5% CO2 for 30 min with Liperfluo and FerroOrange in cell culture medium without FBS. For 7-AAD measurement, after washed by PBS, cells were incubated in binding buffer and 2.5 µM 7-AAD at room temperature. After incubation, cells were examined by flow cytometry within 2 h of staining.

Mouse xenograft model of LLC

Male C57BL/6 mice (6–8 weeks old, ~20 g) were obtained from Beijing Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China). All animals were maintained in air-conditioned and specific pathogen-free animal rooms at 23 ± 2 °C and 45 ± 5% humidity under a 12 h light/dark cycle (8:00 AM to 8:00 PM). Experimental diets and water were provided ad libitum. All mice were maintained in an animal facility and cared for in accordance with the institutional guidelines for animal welfare. All experiments on mice were approved by the Institutional Animal Care and Use Committee of China Agricultural University (RD-202101037-1). Mice were inoculated subcutaneously (s.c.) in the right flank with 5 * 105 LLC tumor cells to establish xenograft model of LLC. The animals bearing cancer xenografts were randomly divided into treatment and control groups.

18β-GA treatment

18β-GA (#G10105) was purchased from Aladdin Ltd, Shanghai, China. For the in vivo experiments, 18β-GA was dissolved in 1% sodium carboxymethyl cellulose (CMC-Na) (vehicle), and stock solutions at 6.6 mg/mL were prepared. The mice were gavage-fed 3 days after tumor inoculation at a daily dose of 18β-GA at 25, 50, 100 mg/kg for 13 consecutive days or its vehicle (n = 5). The doses of 18β-GA were determined based on human and mouse studies [9, 15,16,17,18] along with our dose-finding experiments. Mice doses used in the present study are safe and clinically relevant based on the calculation with the human/mouse conversion table. According to a number of human studies, taking daily doses of 500–1000 mg GA is safe for humans [16,17,18]. Based on the Conversion Table of Animal Doses to Human Equivalent Doses [19], human equivalent dose (HED) (mg/kg) =  Animal does (mg/kg)/12.3 = 100 mg/kg/12.3 = 8.13 mg/kg. The average human body weight is 60 kg, then the daily dose of human = 8.13 * 60 = 487.8 mg. Therefore, the high dose we used in the present study are within the clinically safe range of GA.

Tissue sampling

All mice were killed by cervical dislocation. Tumor tissue was carefully isolated and washed in phosphate-buffered saline (PBS) to remove blood. Separated samples were frozen at −80 °C or fixed in 4% neutral buffered formalin for subsequent determination.

Tumor free fatty acids (FFAs) examination

100 mg tumors were homogenized into 1 mL of ice-cold saline and centrifuged at 3500 r/min for 10 min at 4 °C. FFAs were quantified using kits (Solarbio Cat. #BC0595).

Immunohistochemistry

Formalin-fixed, paraffin-embedded tissues were deparaffinized, boiled for antigen retrieval. Tissue specimens were incubated with antibodies against CD8 (Abcam Cat. #ab217344) or Granzyme B (R&D Systems Cat. #AF1865) overnight at 4 °C. Bound antibodies were detected with biotin-conjugated secondary antibodies and then incubated with an avidin–biotin–peroxidase complex. Visualization was performed using diaminobenzidine (DAB) chromogen. Images were obtained with a VANOX microscope (Olympus, Japan) and analyzed by Image J.

Western blotting

Western blotting (WB) was performed as described previously [20]. Briefly, total protein was extracted from cells. Aliquots of total protein (60 µg) were electrophoresed on sodium dodecyl sulphate polyacrylamide, 10% Tris–HCl gels (Bio-Rad Laboratories, Hercules, CA, USA). The separated proteins were transferred to polyvinylidene difluoride membranes (Bio-Rad Laboratories) and incubated with primary antibodies overnight at 4 °C. Membranes were probed with anti-CD36 (Abcam Cat. #ab133625). The anti-β actin and the second-antibody were purchased from MBL Biotch CO., LTD (Beijing, China).

Statistical analysis

Statistical analysis was performed using SPSS Statistics and drew using GraphPad Prism 8 software (GraphPad Software, San Diego, CA, USA). Descriptive parameters were expressed as mean ± SD. Differences among normally distributed values of 3 experimental groups were analyzed by one-way ANOVA, followed by a Tukey multiple comparisons posttest. Differences between normally distributed values of 2 experimental groups were analyzed by an unpaired Student’s t test. *p < 0.05; **p < 0.01; and ***p < 0.001 compared with control group; #p < 0.05; ##p < 0.01, and ###p < 0.001 compared with AA alone group.