Integrated intracellular and secretome proteomics reveal compartment-specific responses to plasma-treated water solutions combined with doxorubicin in hypopharyngeal carcinoma

Cold atmospheric Plasma-Treated Water Solutions (PTWS) carry reactive oxygen and nitrogen species and may enhance chemotherapy, but their effects on the tumour response remain unclear. We used SWATH-MS and DDA proteomics to characterise intracellular and secreted protein changes associated with Air (richer in nitrite) and oxygen (O₂) (richer in H 2 O 2 ) PTWS combined with doxorubicin (DOXO) in FaDu Head and Neck Cancer cells and HaCaT keratinocytes. This discovery-based study aimed to identify treatment-associated proteomic signatures rather than causal mediators. Proteomic responses were formulation-, cell- and compartment-dependent. In FaDu cells, more than 80% of the secreted proteins modified by PTWS + DOXO were not modified by DOXO alone. Air PTWS + DOXO increased extracellular HSP70, HSP10 and RAB11B, whereas O₂ PTWS + DOXO predominantly reduced secreted protein abundance, with 25 of 33 modified proteins decreased, including cathepsin D, HSP27 and moesin. Both PTWS formulations reduced extracellular PRDX6, while HaCaT cells showed fewer and mainly adaptive changes. Secretome changes were only partly reflected by intracellular proteomics, providing complementary information on the treatment response. Overall, PTWS did not simply amplify the response to DOXO but modified it in a formulation- and cell-dependent manner. The identified secretome signatures provide hypotheses for future functional studies and should not be interpreted as direct evidence of tumour-microenvironment remodelling.