Abstract
Cold atmospheric plasma and more recently, plasma-activated liquids (culture media, water or buffered solutions previously exposed to plasma), are gathering momentum in cancer cells treatment. Nevertheless, in vitro tests show that this novel approach is sometimes less efficient than expected. We here evaluate the mechanisms of action of the plasma-activated PBS and suggest to use electropermeabilization (EP) in combination with the plasma-activated phosphate-buffered saline (PBS), in order to potentiate the cytotoxic effect of the plasma activated liquid. Human multicellular tumor spheroids (MCTS), a three-dimensional cell model, which resembles small avascular tumors, was used to define the optimal treatment conditions for single and dual-mode treatments. MCTS growth, viability, and global morphological changes were assessed by live cell video-microscopy. In addition, the induction of caspases activation, the appearance of DNA damages, and cell membrane permeabilization, as well as the early modifications in the cellular ultrastructure, were examined by immunofluorescence, propidium iodide staining, confocal fluorescence microscopy and transmission electron microscopy, respectively. Altogether, our results show that a combined treatment resulted in an earlier onset of DNA damage and caspases activation, which completely abolished MCTS growth. This report is a proof of concept study evidencing that electropermeabilization greatly potentiates the cytotoxic effect of plasma-activated PBS in vitro in a three-dimensional cancer cell model.
Highlights
Www.nature.com/scientificreports water (PAW) or physiological solutions such as phosphate buffered saline (PBS) or the NaCl saline solution (P-A PBS/NaCl, respectively)[8]
The Plasma-activated PBS (P-A PBS)’s efficiency was monitored: (i) by live cells fluorescence microscopy, which allowed assessing macroscopic morphological changes and cell membrane permeability occurring in HCT 116-green fluorescent protein (GFP) multicellular tumor spheroid (MCTS) over time; (ii) by transmission electron microscopy (TEM), which enabled us to evaluate modifications in MCTS ultrastructure; (iii) by immunofluorescence, after the immunostaining of phosphorylated histone H2A variant H2AX, which allows assessing DNA damages, and (iv) by a luminescent assay, determining the pro-apoptotic potential of the treatment, measuring caspase-3/7 activities in MCTS
The macroscopic appearance and the viability of MCTS were daily monitored with a wide field microscope (Fig. 1)
Summary
Www.nature.com/scientificreports water (PAW) or physiological solutions such as phosphate buffered saline (PBS) or the NaCl saline solution (P-A PBS/NaCl, respectively)[8]. This capacity of pulsed electric field to reversibly permeabilize cell membranes is nowadays used to increase intracellular delivery of therapeutic molecules to cells and tissues This approach enables the administration of lower local or systemic drug doses[17,18,19]. The 3D organization makes cellular spheroids attractive for the evaluation of drug delivery efficiency upon EP34 In this context, the aim of the present study was to evaluate the effect of P-A PBS in a 3D cancer cells model, and to potentiate the cytotoxic effect, by combining P-A PBS treatment with EP. The P-A PBS’s efficiency was monitored: (i) by live cells fluorescence microscopy, which allowed assessing macroscopic morphological changes and cell membrane permeability occurring in HCT 116-GFP MCTS over time; (ii) by transmission electron microscopy (TEM), which enabled us to evaluate modifications in MCTS ultrastructure; (iii) by immunofluorescence, after the immunostaining of phosphorylated histone H2A variant H2AX, which allows assessing DNA damages, and (iv) by a luminescent assay, determining the pro-apoptotic potential of the treatment, measuring caspase-3/7 activities in MCTS
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