NU/NU nude mice were purchased from Hunan Slac Laboratory Animals Co., Ltd. (Changsha, China). Melanoma cells (5 x 10⁶) were injected subcutaneously into the left posterior side of 7-week-old immunodeficient female mice. The tumor growth was monitored by measuring the length (L) and width (W) of the tumor. Tumor volume = 1/2 (length x width2). When the tumor volume reached about 50 mm³, mice were randomly assigned into 8 groups (n = 6) and given intraperitoneal injection of erastin for 20 days. Erastin was dissolved in 5% DMSO + corn oil (C8267, Sigma) in the test tube heated at 37 and gently shaken before use.

NU/NU nude mice were purchased from Hunan Slac Laboratory Animals Co., Ltd. (Changsha, China). Melanoma cells (5 x 10⁶) were injected subcutaneously into the left posterior side of 7-week-old immunodeficient female mice. The tumor growth was monitored by measuring the length (L) and width (W) of the tumor. Tumor volume = 1/2 (length x width2). When the tumor volume reached about 50 mm³, mice were randomly assigned into 8 groups (n = 6) and given intraperitoneal injection of erastin for 20 days. Erastin was dissolved in 5% DMSO + corn oil (C8267, Sigma) in the test tube heated at 37 and gently shaken before use.

To generate murine subcutaneous tumors, melanoma cells (5 x 10⁶ cells per mouse) were injected subcutaneously into the right posterior flanks of 7-week-old immunodeficient nude mice. When tumors reached a volume of approximately 50 mm³, mice were randomly allocated into groups and treated with erastin for 20 days. The erastin was dissolved in 5% dimethylsulfoxide (DMSO) + corn oil (C8267, Sigma).

NU/NU Nude mice were purchased from Charles River (Beijing). To generate murine subcutaneous tumors, melanoma cells (5 x 10⁶ cells per mouse) were injected subcutaneously into the left posterior flanks of 7-week-old immunodeficient female nude mice.

NU/NU Nude mice were purchased from Charles River (Beijing). To generate murine subcutaneous tumors, melanoma cells (5 x 10⁶ cells per mouse) were injected subcutaneously into the left posterior flanks of 7-week-old immunodeficient female nude mice.

All animal experiments were approved by the Ethical Review of Experimental Animals at Central South University. To generate subcutaneous tumors, 2 x 10⁶ control A375 cells or GPX4 KO cells were suspended in 100 ul PBS and injected subcutaneously into nude mice (Shanghai SLAC). Tumor-bearing mice were randomly allocated into groups and treated with vehicle (2% DMSO + 30% PEG300, per day by orally) or lorlatinib (10 mg/kg, per day by orally). Liproxstatin-1 (10 mg/kg) was administrated through intraperitoneal injection per day. Tumors were weighted and photographed on day 18 after treatment. Tumor size were recorded every three days and calculated as [(length x width x width)/2].

Mice (4-5/group) were injected s.c. in the right back flank with 1 x 10⁶ B16 tumor cells. After 7 to 14 days, tumors formed and were collected for flow cytometry analysis. In some mice, tumor growth was monitored till the end point, and CD8-depleting antibodies (200 ug per mice) were injected one day before tumor injection, followed by 4 consecutive injections every 3 days. In B16 melanoma lung metastatic model, mice were injected i.v. with 2 x 10⁵ B16 cells.

In liproxstatin-1 rescue experiment, 5 x 10⁵ B16F10 cells were subcutaneously injected into the right flank of C57BL/6 mice. When the tumor grows to 50 mm³, 100 ug anti-PD-1 antibody (Bio X Cell, USA), 30 mg/kg liproxstatin-1 (MedChemExpress, USA) or both were administered intraperitoneally to each mouse. Anti-PD-1 antibody was administered every 3 days and liproxstatin-1 was administered every day.

In liproxstatin-1 rescue experiment, 5 x 10⁵ B16F10 cells were subcutaneously injected into the right flank of C57BL/6 mice. When the tumor grows to 50 mm³, 100 ug anti-PD-1 antibody (Bio X Cell, USA), 30 mg/kg liproxstatin-1 (MedChemExpress, USA) or both were administered intraperitoneally to each mouse. Anti-PD-1 antibody was administered every 3 days and liproxstatin-1 was administered every day.

In liproxstatin-1 rescue experiment, 5 x 10⁵ B16F10 cells were subcutaneously injected into the right flank of C57BL/6 mice. When the tumor grows to 50 mm³, 100 ug anti-PD-1 antibody (Bio X Cell, USA), 30 mg/kg liproxstatin-1 (MedChemExpress, USA) or both were administered intraperitoneally to each mouse. Anti-PD-1 antibody was administered every 3 days and liproxstatin-1 was administered every day.

A total of 10 female BALB/c nude mice (4-6 weeks old) were supplied by Shanghai SLAC Laboratory Animal Co., Ltd (Shanghai, China). The nude mice were randomly divided into different treatment groups with five mice each, and maintained in a pathogen-free animal facility, followed by subcutaneously injection with 1 x 10⁷ cells/mL M14 cells in 100 uL PBS on the right side to establish a subcutaneous xenograft model.

All animal experiments were approved by the Ethical Review of Experimental Animals at Central South University. To generate subcutaneous tumors, 2 x 10⁶ control A375 cells or GPX4 KO cells were suspended in 100 ul PBS and injected subcutaneously into nude mice (Shanghai SLAC). Tumor-bearing mice were randomly allocated into groups and treated with vehicle (2% DMSO + 30% PEG300, per day by orally) or lorlatinib (10 mg/kg, per day by orally). Liproxstatin-1 (10 mg/kg) was administrated through intraperitoneal injection per day. Tumors were weighted and photographed on day 18 after treatment. Tumor size were recorded every three days and calculated as [(length x width x width)/2].

For primary tumorigenesis assays, NOD-scid Il2rg-/-mice (6-8 weeks old, female) were injected subcutaneously in the left flank with cultured CTCs, and tumors were harvested when they reached 2 centimeters in diameter.