SELECTIVE MODULATION OF THE CELL SECRETOME BY DOXORUBICIN AND COLD PLASMA TREATED WATER SOLUTION IN HEAD AND NECK CANCER CELL MODELS

Aim: cold atmospheric plasma–treated water solutions (PTWS) are reservoir of reactive species able to modulate cellular responses. This study examined whether PTWS combined with doxorubicin (DOXO) alters secretome profiles in HaCaT and FaDu cells under DOXO, DOXO+Air, and DOXO+ O2 conditions. Methods: cells were exposed to DOXO alone or in combination with PTWS (Air or O2). Supernatants were analyzed by DDA proteomics, filtered Unused >1.3), and normalized by removing control proteins. Functional analysis was performed using GO enrichment and semantic clustering. Results: in HaCaT cells, although many proteins were initially identified (up to n = 219), treatment-specific genes were limited, with the highest number in DOXO+PTWS O2 (n = 14), followed by DOXO+Air (n = 12) and DOXO (n = 9). In FaDu cells, a similar number of proteins was identified (up to n = 207), but treatment-specific genes were more abundant, again highest in DOXO+PTWS O2 (n = 30), followed by DOXO+PTWS Air (n = 12) and DOXO (n = 7). GO enrichment analysis indicated predominantly homeostatic processes in HaCaT cells (coagulation, ECM organization, inflammation, migration), whereas FaDu cells showed enrichment in immune, apoptotic, cytoskeletal, phosphorylation, and remodeling pathways. These signatures are consistent with stress‑ and tumor‑associated activation, particularly under plasma conditions. Conclusions: DOXO+PTWS alters the secretome depending on the formulation of PTWS used. HaCaT responses are limited, whereas FaDu shows broader activation, especially after exposure to PTWSO2, indicating greater tumor cell sensitivity to plasma-based treatment.