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Simple Summary  Bladder cancer has a high incidence and mortality. Besides this, currently available therapies for this type of cancer have low efficacy and show considerable adverse effects, urging the need of new therapeutic approaches. Cold Atmospheric Plasma treatment presents itself as a promising alternative, having demonstrated antitumor effects against several types of cancer. The present work arises from a multidisciplinary team, namely, medical doctors and researchers, in an attempt to find new therapeutic strategies to fight bladder cancer. Therefore, our main objective is to evaluate Cold Atmospheric Plasma effects against bladder cancer, as well as the mechanisms by which it exerts its effects. The results obtained demonstrate that Cold Atmospheric Plasma treatment has a promising antitumor effect on bladder cancer, with higher sensitivity for the high-grade cell line. This new approach using Cold Atmospheric Plasma for the treatment of bladder cancer presents enormous clinical benefits, since it is able to selectively treat the tumor tissue, sparing the normal urothelium, with an additional glaring positive economic impact, since it entails a decrease in the cost of therapy in comparison with conventional therapeutic options.    Abstract  Antitumor therapies based on Cold Atmospheric Plasma (CAP) are an emerging medical field. In this work, we evaluated CAP effects on bladder cancer. Two bladder cancer cell lines were used, HT-1376 (stage III) and TCCSUP (stage IV). Cell proliferation assays were performed evaluating metabolic activity (MTT assay) and protein content (SRB assay). Cell viability, cell cycle, and mitochondrial membrane potential (Δψ m ) were assessed using flow cytometry. Reactive oxygen and nitrogen species (RONS) and reduced glutathione (GSH) were evaluated by fluorescence. The assays were carried out with different CAP exposure times. For both cell lines, we obtained a significant reduction in metabolic activity and protein content. There was a decrease in cell viability, as well as a cell cycle arrest in S phase. The Δψ m  was significantly reduced. There was an increase in superoxide and nitric oxide and a decrease in peroxide contents, while GSH content did not change. These results were dependent on the exposure time, with small differences for both cell lines, but overall, they were more pronounced in the TCCSUP cell line. CAP showed to have a promising antitumor effect on bladder cancer, with higher sensitivity for the high-grade cell line.

Cold Atmospheric Plasma, a Novel Approach against Bladder Cancer, with Higher Sensitivity for the High-Grade Cell Line