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

## TL;DR

Cold plasma combined with cisplatin shows promise in bladder cancer treatment by enhancing chemotherapy effects and altering tumor markers.

## Contribution

The study demonstrates plasma's ability to synergistically enhance cisplatin's cytotoxicity and reshape tumor-associated molecular signatures in bladder cancer.

## Key findings

- Plasma and cisplatin combination showed synergy in SCaBER cells and additive effects in RT-112 and T24 cells.
- Hydrogen peroxide was identified as a key mediator of plasma-induced cytotoxicity in SCaBER cells.
- Combination therapy induced cell line-specific changes in tumor markers and cytokine secretion in the TUM-CAM model.

## Abstract

Cold physical plasma (hereafter referred to as plasma) was evaluated for its potential to enhance cisplatin therapy in bladder cancer models. Using the cell lines RT-112, T24, and SCaBER, the plasma–cisplatin combination showed synergistic effects in SCaBER, additive effects in RT-112, and additive to mildly synergistic effects in T24 cells. Plasma and cisplatin displayed opposing monotherapy sensitivity profiles, favoring combination treatment. Hydrogen peroxide was identified as a key mediator of plasma- and combination-induced cytotoxicity in SCaBER cells. In the in ovo tumor chorioallantoic membrane (TUM-CAM) model, plasma and cisplatin monotherapies comparably reduced tumor burden by inhibiting tumor growth, enhancing tumor immunogenicity, and modulating cytokine secretion. By contrast, the combination treatment had limited effects on tumor mass and vascularization over monotherapies but induced distinct, cell line-specific changes in cellular markers, PD-L1, and secretion profile. Overall, these findings support further preclinical evaluation of plasma as an adjunct to cisplatin therapy in bladder cancer.

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: Three bladder cancer cell lines representing distinct entities were used, namely the urothelial carcinoma lines RT-112 and T24, and the squamous cell carcinoma line SCaBER. IC25 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.