Fluorescence microscopic approach for detection of two different modes of breast cancer cell death induced by nanosecond pulsed electric field

Application of nanosecond pulsed electric field (nsPEF) to living systems is a fast-growing research area with great potential for cancer treatment. Here, we have demonstrated a cellular-level approach applying nsPEF having a pulse width of 50 ns and a frequency of 2 kHz to induce the change in intracellular function and dy-namics for two subtypes of breast cancerous cells, that is, estrogen and progesterone-positive (ER+ and PR+) and human epidermal growth factor 2 (HER2) negative MCF-7 cells and highly aggressive drug resistance triple -negative MDA-MB-231 cells. Microscopic monitoring of the field effects in real-time or before and after the application of nsPEF indicated that nsPEF induced different field effects on intracellular function and dynamics in MCF-7 and MDA-MB-231 cells and activated two different modes of cell death, that is, apoptosis in MCF-7 cells and necrosis (or necroptosis) in MDA-MB-231 cells. These two different modes of nsPEF-induced cell death were confirmed based on the following microscopic measurements: 1) autofluorescence intensity and lifetime imaging of nicotinamide adenine dinucleotide (NADH); 2) production of reactive oxygen species (ROS); 3) change in mitochondrial membrane potential; 4) morphological alternation of cells; 5) imaging of phosphatidyl serine (PS) externalization by Annexin V; 6) cell viability. The present finding of nanosecond pulsed electric field-induced apoptosis and necrosis (or necroptosis) in subtypes of breast cancerous cells provides valuable information that the mechanism of field-induced cell death depends on the subtype of cancerous cells. It will be helpful for further development and optimization of field-induced cancer therapy.