A novel transported cold atmospheric plasma platform for induction of apoptosis in breast cancer stem cells

Abstract

Breast cancer stem cells (BCSCs) promote tumour recurrence, metastasis, and therapeutic resistance through enhanced anti-apoptotic signalling and impaired p53 function. Cold atmospheric plasma (CAP) is an emerging anticancer modality capable of generating reactive oxygen and nitrogen species (RONS) that disrupt redox homeostasis and activate apoptosis; however, its specific molecular effects on BCSCs remain unclear. Here, we developed a transported cold atmospheric plasma (T-CAP) system that delivers RONS through a two-stage plasma-transfer pathway using helium or argon carrier gases. MCF7-derived BCSCs were cultured in a three-dimensional Matrigel scaffold and exposed to T-CAP for 60, 120, or 180 seconds. Apoptosis was evaluated by Annexin V/PI flow cytometry, MTT viability assays, and semi-quantitative RT-PCR of Bax, Bak, Bcl-2, Bcl-xL, and p53, supported by transmission electron microscopy (TEM). Plasma-generated species were characterized by optical emission spectroscopy. T-CAP significantly increased Bax, Bak, and p53 expression while downregulating Bcl-2 and Bcl-xL in a time-dependent manner, shifting the balance toward a pro-apoptotic phenotype. Flow cytometry confirmed substantial increases in apoptotic populations, with helium-based T-CAP producing stronger effects than argon. Correspondingly, cell viability declined in MTT assays, and TEM imaging revealed classic apoptotic features, including chromatin condensation, apoptotic body formation, and lysosomal activation. These outcomes correlated with the higher RONS output of helium T-CAP while maintaining relatively low electrical parameters at the treatment site (<0.4 kV and <2 W). Collectively, these findings demonstrate that T-CAP induces apoptotic responses in BCSCs accompanied by reactivation of p53 signalling and modulation of Bcl-2 family proteins, highlighting its potential as a strategy for inducing apoptosis in apoptosis-resistant breast cancer stem cells.