Combination of Plasma-Treated Water and Doxorubicin Drives Compartment-Specific Proteome Reorganization in Head and Neck Cancer Cells

Cold Atmospheric Plasma-Treated Water Solutions (PTWS) are reservoirs of reactive species with anticancer activity. Their combination with doxorubicin (DOXO) enhances cytotoxicity and modulates key cellular processes. This study aimed to compare intracellular and extracellular proteomic profiles in HaCaT and FaDu cells exposed to DOXO alone or in combination with distinct PTWS, to investigate compartment-specific proteome reorganization. HaCaT and FaDu were treated with DOXO alone, DOXO + Air PTWS, or DOXO + O₂ PTWS. Lysates and supernatants were analysed by SWATH-MS and DDA proteomics. Both datasets were normalized by removing control proteins. Functional annotation was performed using GO enrichment and semantic clustering. SWATH-MS revealed treatment-dependent differences between HaCaT and FaDu secretomes. A total of 782-785 proteins were quantified per condition, and significantly modulated proteins (p < 0.05) were retained. Under DOXO alone, HaCaT (n = 19) and FaDu (n = 20) showed no overlap. Plasma treatment amplified divergence, with minimal shared proteins under both Air and O₂ conditions (n = 1). HaCaT showed a predominantly downregulated profile, whereas FaDu exhibited a stronger response, with upregulation of proteins associated with oxidative stress and tumor progression (PRDX6, AKTS1). Integration with lysate proteomics revealed compartment-specific organization. Lysates contained more proteins than secretomes (HaCaT up to 450 vs 9–14; FaDu up to 504 vs 7–30), with minimal overlap. Only KRT1 (HaCaT) and H2AJ (FaDu) were shared between compartments. Lysates were enriched in intracellular processes such as DNA/RNA regulation, oxidative stress and cytoskeletal organization, whereas secretomes were associated with migration, adhesion and extracellular remodeling. HaCaT and FaDu exhibit highly divergent and compartment-specific proteomic responses. PTWS treatments preferentially activate tumor-associated pathways in FaDu while inducing adaptive responses in HaCaT. DOXO + O₂ PTWS elicited the strongest and most selective modulation in FaDu, supporting its potential as a targeted anticancer strategy.