Synergistic Toxicity of Cold Gas Plasma and Cisplatin in Bladder Cancer Cells

BACKGROUND/OBJECTIVES: Bladder cancer remains a therapeutically challenging malignancy due to high recurrence rates, progression to muscle-invasive disease, and frequent resistance to cisplatin-based chemotherapy. Cold physical plasma (hereafter referred to as plasma) has emerged as a locally applicable modality that generates reactive oxygen species (ROS) and shows preclinical antitumor activity, offering a potential strategy to enhance cisplatin efficacy while enabling dose reduction. Here, we investigated combination treatment with cisplatin and argon plasma generated by the clinically approved kINPen jet in human bladder cancer models. METHODS: values for plasma and cisplatin monotherapy were established by resazurin assay and used to design combination regimens. Treatment interactions were quantified by coefficient of drug interaction (CDI) analysis and monitored kinetically by long-term live-cell imaging. Plasma-derived ROS were measured in PBS and DMEM, and their functional relevance was assessed in SCaBER cells using catalase and N-acetylcysteine. In ovo validation was performed in the tumor chorioallantoic membrane (TUM-CAM) model, where tumor mass, vascularization, cellular marker expression, and cytokine secretion were analyzed. RESULTS: Plasma and cisplatin exhibited opposing monotherapy sensitivity profiles across cell lines, creating a favorable basis for combination treatment. CDI analysis revealed clear synergy in SCaBER at intermediate cisplatin concentrations, additive effects in RT-112, and additive to mildly synergistic effects in T24. ROS profiling and scavenger experiments identified hydrogen peroxide as a key mediator of plasma and plasma-cisplatin cytotoxicity in SCaBER. In the TUM-CAM model, plasma and cisplatin monotherapies showed notable antitumoral potential. At the same time, plasma-cisplatin combination therapy elicited only modest effects on tumor growth and vascularization compared to monotreatments but induced distinct, cell line-specific alterations in cytokine and marker expression. CONCLUSIONS: These findings demonstrate that plasma can potentiate cisplatin cytotoxicity in bladder cancer cells and reshape tumor-associated molecular signatures, supporting further optimization and preclinical evaluation of plasma-cisplatin combination therapy.