PEGylated liposomal fluopsin C triggers cuproptosis and ferroptosis pathways and suppresses 3D tumor spheroid growth in NCI-H460 cells

Copper-based metallodrugs are promising anticancer agents, but their toxicity limits clinical translation. This study evaluated the toxicological and molecular effects of PEGylated Fluopsin C (PEG-FlpC) in in vitro human non-small-cell lung carcinoma (NCI-H460) cells cultured as 2D monolayer and 3D spheroids. Cells were exposed to PEG-FlpC at its IC50 (1.0 µM) for 24 h. Flow cytometry revealed G₁-phase arrest and non-apoptotic cell death. PEG-FlpC markedly suppressed spheroid growth, with 2 × IC50-treated spheroids growing only 58.1% compared to 131.6% in the control. Ultrastructural analyses showed mitochondrial clustering and swelling, ER dilation, and cytoplasmic vacuolization. Gene profiling demonstrated downregulation of TP53 and BECN1, with upregulation of TP73 and SQSTM1, consistent with autophagy blockade and stress activation. PEG-FlpC modulated ferroptosis-related (GPX4, SLC7A11, and TFRC) and cuproptosis-related (ATP7B, MTF1, DLAT, and CDKN2A) genes. Pharmacological inhibition confirmed a predominantly copper-mediated mechanism, as TTM (10 µM) restored 77.7% of metabolic activity. Combined inhibition with TTM and Fer-1 (1 µM) further increased viability to 89.4%, indicating that PEG-FlpC induces non-apoptotic regulated cell death through an interconnected cuproptosis–ferroptosis mechanism associated with mitochondrial dysfunction.