Abstract: Background/Objectives: Cold atmospheric plasma (CAP) has shown a strong anticancer effect on a variety of tumors, presenting a new approach for the effective treatment of oral squamous cell carcinoma (OSCC), one of the most prevalent malignant neoplasms with a high mortality rate. Here, we aimed to comprehensively investigate the antitumor potential of two approaches of CAP treatment on both two-dimensional and three-dimensional OSCC cell line models, as well as to analyze whether plasma treatment enhances the sensitivity of OSCC to chemotherapy. Methods: An in-house designed plasma needle, with helium as a working gas, was used to treat the SCC-25 cell line directly or indirectly via plasma-treated medium (PTM). The antitumor effect of CAP was assessed by measuring cell viability, apoptosis, adhesion, and migration. In addition, the combined effect of PTM and cisplatin was analyzed in SCC-25 tumor spheroids, as a more complex and reliable in vitro model. Results: Both plasma treatments showed time-dependent antitumor effects affecting their viability, adhesion, and migration. The rate of apoptosis was higher after incubation with PTM and is mediated by the intrinsic pathway. By utilizing the 3D spheroid carcinoma model, we confirmed the antitumor potential of CAP and additionally demonstrated an increased chemosensitivity of PTM-treated carcinoma cells. Conclusions: The results of our study illustrate a promising avenue for the application of CAP as a therapeutic option for OSCC, either as a standalone treatment or in combination with cisplatin.
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