In vitro study of cold atmospheric plasma–induced proliferation inhibition and morphological changes in head and neck carcinoma cell lines

Objectives: The use of cold atmospheric plasma (CAP) has been growing in medical field. Since limited data exist on the influence of CAP exposure on head and neck cancer (HNC) cells, we aimed to investigate in vitro its impact on proliferation and morphology of HNC (HSC2, HSC3, and FaDu) cells, as well as healthy gingival fibroblasts (hGF). Methods: A CAP gas air jet was applied directly on the HSC2, HSC3, FaDu and hGF cells for 30 and 60 s; doxorubicin has been used as positive control. Inhibition of cell proliferation at different time points (24, 48, and 72 h) was investigated through MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium]. Morphological alterations were observed using scanning electron microscope (SEM) and transmission electron microscope (TEM). Data were statistically analyzed (p < 0.05). Results: CAP exposure of 60 s showed the highest proliferation inhibitory effects in all investigated cancer cell lines (p < 0.001). While HSC2 had the highest proliferation inhibition at 24 h, HSC3, and FaDu exhibited a positive, time-dependent inhibitory effect (24 <48 <72 h) after CAP exposure. Interestingly, effects of CAP in the hGF were minimal. SEM observations revealed morphological changes in HNC cells treated with CAP at 30 and 60 s. Moreover, TEM showed stressed conditions in the survived HNC cells at 48 h after CAP exposure demonstrated by the presence of multilamellar and multi-vesicular bodies. Conclusions: CAP treatment induced proliferation inhibition, morphologic changes and stressed conditions in HNC cell lines (HSC2, HSC3, and FaDu), while demonstrating limited effects on hGF cells. Clinical Significance: This work characterized early cellular responses of HSC2, HSC3, and FaDu to CAP exposure, including proliferation, and morphology, as foundational data before progressing to more clinically representative models.