080 | Monocytes succumb to protect head and neck cancer cells from plasma-treated water solutions cytotoxicity

Giuseppe Natali¹, Anna Martina Battaglia², Emanuele Giorgio², Lavinia Petriaggi², Alessandro Antonelli³, Cristiana Galeano², Amerigo Giudice³, Eloisa Sardella⁴, Vittoria Perrotti⁵, Flavia Biamonte² | ¹Department of Health Sciences, Magna Graecia University of Catanzaro, Italy; ²Department of Experimental and Clinical Medicine, Magna Graecia University of Catanzaro, Italy; ³Department of Health Science, School of Dentistry, Magna Graecia University of Catanzaro, Italy; ⁴CNR-Istituto di Nanotecnologia CNR-NANOTEC UoS Bari, c/o Dipartimento di Chimica, Università degli Studi di Bari Aldo Moro, Bari, Italy; ⁵Department of Innovative Technologies in Medicine & Dentistry, “G. d’Annunzio” University of Chieti-Pescara, Chieti, Italy.

Plasma-treated water solutions (PTWS), enriched with long-lived reactive oxygen and nitrogen species (RONS), are emerging as a tumor-selective therapeutic strategy. Here, FaDu and SAS head and neck cancer (HNC) cells, as well as HaCaT healthy epithelial cells, were treated with PTWS air 20’and oxy 20’ for 24h. First, we found that SAS cells were the most sensitive, as treatment with both PTWS formulations induced ~65% cell death (~35% late apoptotic and ~30% non-apoptotic events). This was associated with a 2-fold increase of ROS levels, along with ~27% and ~56% of lipid peroxidation upon PTWS-air 20′ and PTWS-oxy 20′, respectively. FaDu cells displayed an intermediate cytotoxicity of ~20% with PTWS-air 20′ and ~50% with PTWS-oxy 20′, characterized by partially early apoptotic and non-apoptotic events. Consistently, ROS levels showed only a modest increase under both PTWS formulations, along with <10% of lipid peroxidation. Conversely, HaCaT cells showed low PTWS-induced cytotoxicity (<30% for both formulations), with mild ROS induction insufficient to trigger lipid peroxidation. Therapeutic efficacy of PTWS could be strongly influenced by the tumor microenvironment (TME), where innate immune cells, such as monocytes, shape stress-induced cytotoxicity. To this, SAS and FaDu cells were co-coltured with THP-1 monocytes. THP-1 cells alone displayed partial susceptibility to PTWS-air 20′ (18%) and -oxy 20′ (59%). Notably, in co-colture, THP-1 reached ~99% mortality, accompanied by a marked decline in SAS cell death (~50%), by preferentially attenuating apoptosis. This was associated with a reduction of lipid peroxidation to undetectable levels. In FaDu cells, THP-1 co-culture caused a slight decrease in overall mortality, an arrest in early apoptosis, along with reduced lipid peroxidation to ~1%. Overall, these data identify monocytes as key determinants of HNC responsiveness to PTWS treatment and highlight the need for mechanistic insight into the use of PTWS in vivo.