Tumor-repopulating cells (TRCs) evade ferroptosis via PCK2-dependent phospholipid remodeling. TRCs, which have stem-cell-like characteristics, resist chemotherapy and radiotherapy by reducing ferroptosis sensitivity. Mechanistically, mitochondrial metabolic kinase PCK2 phosphorylates and activates ACSL4, driving ferroptosis-associated phospholipid remodeling. TRCs downregulate PCK2 to achieve a structural ferroptosis-resistant state. PCK2-pACSL4(T679) plays a critical role in multiple preclinical models, and higher PCK2 and pACSL4(T679) levels correlate with better response to chemotherapy and radiotherapy and lower distant metastasis in nasopharyngeal carcinoma (NPC) cohorts. Ferroptosis is characterized by iron-dependent lipid peroxidation. Accumulating evidence suggests that ferroptosis plays a critical role in tumor suppression. Tumor suppressor genes and immune factors are involved in enhancing or inducing ferroptosis, leading to the suppression of tumor progression. Chemotherapy, radiotherapy, and immunotherapy can induce ferroptosis. Stem-cell-like cancer cells (SCLCCs), which are self-renewing and highly tumorigenic, can repopulate tumors and are resistant to multiple treatments, mediating tumor recurrence. The resistance of SCLCCs toward various treatments is primarily mediated by avoiding programmed death processes to escape from predetermined death. However, whether SCLCCs avert ferroptosis to mediate therapy resistance remains unclear. The degree of phospholipid (PL) unsaturation is a critical factor in determining the susceptibility of cells to ferroptosis. Acyl-CoA synthetase long chain family member 4 (ACSL4) catalyzes the production of polyunsaturated fatty acid (PUFA)-CoA. Activated PUFA is further integrated into PLs catalyzed by membrane-bound O-acyltransferases, particularly lysophosphatidylcholine acyltransferase 3 (LPCAT3), which subsequently enters the cell membrane. Post-transcriptional modifications, such as phosphorylation of ACSL4, are crucial for regulating enzymatic activity, and different kinases or modification sites may lead to opposite effects. Interestingly, a special class of kinases, such as phosphoglycerate kinase 1 (PGK1) or pyruvate kinase M2 (PKM2), which typically act as metabolic enzymes to mediate the phosphorylation of small-molecule metabolites, can also function as protein kinases, thereby creating strong crosstalk between metabolism and signal transduction. In this study, we used a biomechanical forces-based three-dimensional (3D) soft fibrin gel culture system to obtain TRCs and further explored whether the resistance of TRCs to chemotherapy and radiotherapy was related to ferroptTumor-repopulating cells (TRCs) evade ferroptosis via PCK2-dependent phospholipid remodeling. TRCs, which have stem-cell-like characteristics, resist chemotherapy and radiotherapy by reducing ferroptosis sensitivity. Mechanistically, mitochondrial metabolic kinase PCK2 phosphorylates and activates ACSL4, driving ferroptosis-associated phospholipid remodeling. TRCs downregulate PCK2 to achieve a structural ferroptosis-resistant state. PCK2-pACSL4(T679) plays a critical role in multiple preclinical models, and higher PCK2 and pACSL4(T679) levels correlate with better response to chemotherapy and radiotherapy and lower distant metastasis in nasopharyngeal carcinoma (NPC) cohorts. Ferroptosis is characterized by iron-dependent lipid peroxidation. Accumulating evidence suggests that ferroptosis plays a critical role in tumor suppression. Tumor suppressor genes and immune factors are involved in enhancing or inducing ferroptosis, leading to the suppression of tumor progression. Chemotherapy, radiotherapy, and immunotherapy can induce ferroptosis. Stem-cell-like cancer cells (SCLCCs), which are self-renewing and highly tumorigenic, can repopulate tumors and are resistant to multiple treatments, mediating tumor recurrence. The resistance of SCLCCs toward various treatments is primarily mediated by avoiding programmed death processes to escape from predetermined death. However, whether SCLCCs avert ferroptosis to mediate therapy resistance remains unclear. The degree of phospholipid (PL) unsaturation is a critical factor in determining the susceptibility of cells to ferroptosis. Acyl-CoA synthetase long chain family member 4 (ACSL4) catalyzes the production of polyunsaturated fatty acid (PUFA)-CoA. Activated PUFA is further integrated into PLs catalyzed by membrane-bound O-acyltransferases, particularly lysophosphatidylcholine acyltransferase 3 (LPCAT3), which subsequently enters the cell membrane. Post-transcriptional modifications, such as phosphorylation of ACSL4, are crucial for regulating enzymatic activity, and different kinases or modification sites may lead to opposite effects. Interestingly, a special class of kinases, such as phosphoglycerate kinase 1 (PGK1) or pyruvate kinase M2 (PKM2), which typically act as metabolic enzymes to mediate the phosphorylation of small-molecule metabolites, can also function as protein kinases, thereby creating strong crosstalk between metabolism and signal transduction. In this study, we used a biomechanical forces-based three-dimensional (3D) soft fibrin gel culture system to obtain TRCs and further explored whether the resistance of TRCs to chemotherapy and radiotherapy was related to ferropt