Effects of helium cold atmospheric plasma (CAP) on HT-29 cancer stem cell spheroid

Colorectal cancer (CRC) recurrence and therapeutic resistance are largely attributed to cancer stem cells (CSCs), a subpopulation of tumor cells characterized by self-renewal and differentiation capacities. These cells exhibit enhanced antioxidant defenses that maintain redox homeostasis and contribute to resistance against conventional therapies. Cold atmospheric plasma (CAP), a partially ionized gas that generates reactive oxygen and nitrogen species (RONS), has emerged as a promising strategy to exploit redox vulnerabilities in cancer cells; however, its effects on colorectal CSCs remain poorly defined. In this study, CSC-enriched spheroids derived from HT-29 cells were exposed to a helium-based plasma jet for 30 to 240 s. CAP-induced hydrogen peroxide (H₂O₂) generation was quantified, cell viability was assessed using the MTT assay, self-renewal capacity was evaluated via sphere-forming efficiency, and the expression of stemness-associated genes (SOX2, OCT4, and NANOG) was analyzed by qPCR. CAP treatment generated H₂O₂ in a time-dependent manner across all tested media, with DMEM/F12 supplemented with 10% FBS exhibiting the highest accumulation. Spheroid viability declined proportionally with increasing exposure duration. Notably, CAP markedly suppressed self-renewal capacity. Control spheroids reached a diameter of 247 μm, whereas 240-second CAP exposure reduced the diameter to 87 μm (p < 0.0001), corresponding to a 65% reduction. At the molecular level, OCT4 expression increased by twofold (p < 0.01), while SOX2 and NANOG expression levels remained unchanged. Overall, CAP limits regrowth of HT-29 CSC-enriched spheroids in a time-dependent manner, consistent with ROS-associated oxidative stress, as reflected by H₂O₂ accumulation. The stemness transcriptional response was not uniform with increased OCT4 expression, but unchanged SOX2 and NANOG levels. These findings warrant further translational investigation, including comprehensive reactive-species profiling.