# Morphological Changes in H1299 Human Lung Cancer Cells Following W-Band Millimeter-Wave Irradiation
## 4 Discussion  
Cancer is characterized by an excessive and uncontrolled division of abnormal malignant cells that display morphological, proliferative, and functional heterogeneity [23]. Cell size is an important morphological criterion characterizing the physiological status of a cell. The majority of animal cells are 10-20 \(\upmu\)m in diameter and rarely vary more than two folds outside of this range, suggesting that the mechanism for cell size regulation is highly conserved [31]. Changes in H1299 cell morphology following exposure to 75-110 GHz MMW for 2- and 4-min irradiation regimes, respectively, were examined in this study. Past investigations of MMW irradiation effects on normal and cancer cells reported distinct non-thermal biological effects using discrete narrow range(s) of frequency with very low energy [32; 33]. The majority of the H1299 human lung cancer cells ranged from 1000 \(\upmu\)m\({}^{2}\) to 2500 \(\upmu\)m\({}^{2}\) in size before MMW treatment. In response to 2- and 4-min exposure regimes, the cells showed significant changes in the size, circularity, and Feret's diameter. Two morphologically distinct cell groups were found in the post-irradiation observation period lasting up to 14 days. Based on a comparison with sham-untreated samples, these morphological criteria identified two distinct groups: One with a smaller size (500-1000 \(\upmu\)m\({}^{2}\)) and more oval shape characterizing pro-apoptotic cells and a second group comprised of cells bigger in size (2500 to 4000 \(\upmu\)m\({}^{2}\)) with a flattened morphology characteristic of non-dividing senescent cells. The changes in morphology were observed on the same day of MMW exposure treatment (short-term effects), as well as over 7-14 days post-irradiation (long-term effects). The short-term effects may result from regular stress responses following irradiation. The long-term effects arise specifically as a result of MMW irradiation and were observed to be retained over the duration of the experiment(s). Senescent cells are known to present an enlarged phenotype as compared to non-senescent cells [30]. This suggests that the dramatic increment of cell size observed under the 2-min irradiation regime indicates a population shift towards a higher number of senescent cells induced by MMW irradiation. In contrast, the results from the 4-min exposure regime, which delivered a higher dose of MMW energy, indicated an induction of apoptosis as well as senescence. Prior to exposure, a small proportion of the cell population comprised mitotically arrested huge polyploid cells whose numbers increased following irradiation. Recent publications demonstrated that they may form due to perturbations of the molecular mechanisms controlling replication and cell division [34; 35]. In H1299 cells, they were represented by two phenotypes: One with a large single nucleus and the other with multinucleated cells (MNCs) containing up to 11 nuclei. A relatively small proportion of the senescent group (following MMW exposure) were observed to be huge polyploid cells, as it has previously been reported that they generally constitute a part of the cancer cell population responsible for giving rise to drug resistance in anticancer treatments [36]. The 2-min exposure regime induced the development of large cells gradually over 14 days of observation while the 4-min regime induced such effects immediately and over 7 and 14 days following exposure. This indicates that the effects are dose dependent, explaining the absence of enlarged cells immediately following the 2-min exposure regime and induction of both apoptosis and senescence in the 4-min exposure regime. The experiments show that without any treatment, the morphological heterogeneity presented by different size groups is maintained. Following exposure to MMW treatment, the number of cells in different subgroups changed significantly. MMW exposure at an incident power density (IPD) of 20 mW/cm\({}^{2}\) corresponding to the ICNIRP safe public exposure standards results in thermal stress on cells [37].