3D Cancer Cell Model: A New Tool to More Accurately Evaluate the Biological Effect of Cold Plasma

Cold atmospheric plasma (CAP) has emerged as a promising anticancer strategy, combining non-thermal physicochemical properties with selective biological activity. Generated at atmospheric pressure and near-physiological temperatures, CAP produces a complex mixture of reactive oxygen and nitrogen species (RONS), charged particles, UV photons, and transient electric fields capable of disrupting redox homeostasis and activating regulated cell death pathways in cancer cells. Since its first application in oncology, extensive in vitro studies have demonstrated CAP efficacy across a wide range of tumor types, including highly aggressive and therapy-resistant cancers. Most mechanistic insights into CAP activity have been derived from two-dimensional (2D) monolayer cultures, which have revealed key processes such as oxidative stress induction, DNA damage, metabolic rewiring and apoptosis. However, 2D models fail to recapitulate essential features of solid tumors, including three-dimensional architecture, extracellular matrix interactions, and diffusion gradients that critically influence treatment responses. Recent advances increasingly rely on three-dimensional (3D) cancer models, such as multicellular spheroids and matrix-based constructs, which better mimic tumor microenvironmental complexity. Studies using these systems consistently show that CAP and plasma-activated media reduce tumor viability, inhibit growth, and induce oxidative stress-driven cell death, while uncovering resistance mechanisms and dose-dependent effects not observed in 2D cultures. This review highlights the pivotal role of advanced 3D models in improving the predictive value and translational relevance of CAP-based anticancer therapies.