## 4 Discussion  
The aim of the present study was to investigate the effect of low-intensity W-band (75-105 GHz) MMW irradiation on heterogeneous, aggressive, drug-resistant human H1299 lung cancer cells as compared to normal non-tumorigenic MCF-10A epithelial cells. Each cell sample was irradiated once for 2, 4, or 10 min and the change in cell morphology and viability was followed for up to 14 days. Irradiation of H1299 cells for 2 and 4 min led to observable changes in the cell and nucleus morphology, significantly increasing cell mortality and senescence over 7-14 days following MMW exposure. The effects were found to be specific and irreversible in the MMW treated H1299 cells and were not observed in the control group of non-irradiated (control) H1299 cells. In contrast non-tumorigenic immortalized MCF10A cells did not present the same MMW treatment effects even after prolonged irradiation for 16 min (Supplementary Figure S1), over the 14 days of experimental observation. Although cell death and senescence both increased in H1299 cell populations during the experiment, their temporal kinetics were different. Cell mortality peaked seven days after irradiation, while senescence peaked on Day 14 for 2 and 4 min of irradiations. Cell deaths occurred likely due to the inability of MMW exposed cells to repair themselves after irradiation, while some damaged cells survived and formed an aged cell population. Such senescent cells are reported to survive for a long time and arrest the apoptotic stimuli of the physiological cycle [26]. These two different effects possibly originate from activation and/or inhibition of different physiological mechanisms involving tumor development [2]. The effects of irradiation on cell death and senescence were confirmed by observations of non-thermal morphological changes previously reported [19]. Changes in cell shapes occurred parallel to the changes in cell size following MMW exposure. Decrease in the cell size was in accordance with the increase in their circularity and cell diameter and vice versa. In the current study, changes in nuclear shape and size were also observed after MMW treatment. Such physical changes indicate the formation of two functionally distinct cell groups: pro-apoptotic and senescent. The pro-apoptotic cells present decreased cell and nuclei sizes with increased circularity, in addition to fragmented nuclei indicating internal damage. The pro-senescence cell phenotype shows an increase in both cell and nuclei sizes. They exhibit flattened forms containing \"puffed\" nuclei with kidney-like shapes [27]. A small proportion of the H1299 cell population comprised mitotic and huge polyploid cells whose numbers increased following irradiation, possibly due to nuclear changes. Reports suggest their formation arising from perturbations of the molecular mechanisms that control replication and cell division [28; 29]. In H1299 cells they were represented by two phenotypes: one with a large single nucleus and the other-with multinucleated cells (MNC) containing up to 11 nuclei. These cells were positively stained with the \(\beta\)-galactosidase (SA-\(\beta\)-Gal) senescence marker and had an ability to divide although the cytoplasm exhibited no division. Therefore, these cells are suspected to have been resistant to 2 and/or 4 min irradiation (which delivered lower energy dosage as compared to the terminal cytotoxic effect of 10 min regime); likely to be the source of the cell population that survived over 7-14 days. Studies similarly report that polyploidy cells were more resistant to chemotherapeutic agents than mononuclear cells (MNCs) [29]. Further, MNCs undergo asymmetric division (producing mononuclear cells) and self-renewal in vitro and in vivo; with a single cell capable of generating orthotropic subcutaneous tumors (composed mainly of mononuclear cells) giving rise to spontaneous lung metastases in nude mice. However, the 10 min exposure regime successfully terminated these multinuclear as well as the mononuclear H1299 cells.