Selective Secretome Modulation by PTWS and Doxorubicin in Hypopharyngeal Cancer Cells vs Keratinocytes

Objective: 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 the secretome profiles of keratinocytes (HaCaT) and hypopharyngeal cancer (FaDu) cell line exposed to DOXO alone or in combination with distinct PTWS in order to identify the molecular pathways modulated by each treatment condition. Methods: HaCaT and FaDu were treated with DOXO alone or in combination with PTWS generated using air or O₂ for 10 minutes and comparative analysis was performed for each condition (DOXO, DOXO+ Air PTWS, DOXO + O2 PTWS). Cell culture supernatants were analysed by SWATH-MS proteomics and normalized by removing control proteins. Data-Dependent Acquisition (DDA) datasets were used for functional annotation through Gene Ontology (GO) enrichment and semantic similarity clustering. Results: Comparative SWATH proteomic analysis revealed pronounced and treatment-dependent differences in secretome composition between HaCaT and FaDu cells. A total of 782-785 proteins were quantified per condition, and only significantly modulated proteins (p < 0.05) were retained for downstream analysis. Under DOXO, a comparable number of differentially expressed proteins was identified in HaCaT (n = 19) and FaDu (n = 20), with no shared proteins, indicating completely divergent responses. HaCaT showed upregulation of proteins associated with adaptive stress - RL17 and C1TC - whereas FaDu exhibited modulation of distinct targets, including F10A1, involved in invasiveness and migration. Under DOXO + Air PTWS, HaCaT showed fewer differentially expressed proteins (n = 17) than FaDu (n = 29), with only one shared protein, MYH9, a cytoskeletal regulator involved in cell motility, which displayed opposite regulation in HaCaT and FaDu. While HaCaT proteins’ spectra was predominantly downregulated, FaDu showed upregulation of proteins such as MATR3 and RCC2, again involved in cytoskeleton remodelling. Under DOXO + O₂ conditions, HaCaT exhibited a weaker response (n = 13) compared to FaDu (n = 33), again with only one shared protein, TBG1, involved in microtubule organization, which was consistently downregulated in both HaCaT and FaDu. In addition, FaDu displayed strong upregulation of multiple proteins, including TRUB1, F10A1, PRDX6 and AKTS1 linked to oxidative stress and apoptosis pathways, whereas HaCaT showed a predominantly downregulated profile. Conclusion: Direct comparison between HaCaT and FaDu revealed divergent and treatment-specific secretome profiles with minimal overlap. HaCaT predominantly exhibited downregulation of factors involved in protein homeostasis and translation (PSMD7, EIF3B, EF1A1), consistent with a controlled adaptive response. In contrast, FaDu displayed upregulation of proteins associated with tumor-related pathways, including oxidative stress (PRDX6), PI3K/AKT survival signaling (AKTS1), cytoskeletal remodeling (RCC2, MOES) and extracellular proteolysis (CATD), indicating activation of adaptive and pro-survival mechanisms. DOXO + O2 PTWS induced the strongest and most selective modulation in FaDu secretome, suggesting potential as a selective anticancer adjuvant, whereas DOXO + Air PTWS elicited a milder response mainly related to cytoskeletal remodeling. Overall, plasma-based treatments preferentially activated tumor-associated pathways in FaDu cells while promoting adaptive homeostatic mechanisms in HaCaT, supporting a possible selective therapeutic effect.