# Morphological Changes in H1299 Human Lung Cancer Cells Following W-Band Millimeter-Wave Irradiation
## 3 Results
### MMW Irradiation Increases Cell Circularity and Feret's Diameter of H1299 Cancer Cells  
In order to ascertain that MMW irradiation induced apoptosis and senescence as suggested by the results above, further stringent parameters were quantified. Specifically, cell circularity and Feret's diameter were measured to determine the extent of the said effects. Circularity values ranging from the least circular shape (value 0) to a perfect circle (value 1) are used to denote the degree of dimensional roundness of an object. Increased cell circularity resulting from cytoplasmic shrinkage and cell fragmentation is a hallmark of apoptosis [27; 24]. The majority (80%) of untreated control cells presented circularity values between 0.5 and 0.8 (Figure 4a). Following MMW exposure, the circularity of irradiated cell populations increased by 1.5 times as compared with that of control untreated cells. The 2-min irradiation regime led to a population shift towards a more circular shape (0.8-0.9) on Day 1 (Figure 4b). Seven days of physiological incubation following exposure in this regime presented cell populations split into two groups. One group retained the circular form (0.8-0.9), indicating apoptotic or programmed cell death (PCD) cells, and another a less circular form (0.3-0.5) than untreated cells, likely to be fragments of PCD cells that lost their normal form and size. Interestingly, the split in the population was retained even after 14 days following irradiation. The 4-min irradiation regime resulted in a similar but more marked trend of the population shift (Figure 4c). As these effects persisted over 7 to 14 days of the post-irradiation period and were not observed in the untreated control cells, the changes in cell circularity were concluded to be a specific response to MMW irradiation. Feret's diameter (FD) measured as the longest straight line between two points on the periphery of a cell is an important morphological marker of cells undergoing senescence [29]. Larger FD corresponds to longer cellular extensions (i.e., protrusions), and enlarged structurally aberrant cells characteristic of senescence [30]. The FD of non-irradiated cells did not change over the experimental time course (Figure 4d). The 2-min exposure regime led to a significant increase of FD over 7 days of physiological incubation following irradiation (Figure 4e) and was reversed after 14 days. In contrast, the 4-min irradiation regime resulted in an FD increase over 14 days and not 7 days (Figure 4f). Percentages higher than 100% are accountable to the splitting and reseeding of cells over the period of observation in order to maintain constant confluence rates (30%) for stringency of analyses (see methods). These results demonstrate that a significant number of cells changed their shapes and sizes in an energy (dose)-dependent manner as a specific response to MMW irradiation. Trigonal, flattened, and adherent cells of the control groups changed to oval-shaped cell bodies with long protrusions following the MMW exposure treatment. Figure 4: Distributions of cell circularity (**A**–**C**) and Feret’s diameter (**D**–**F**) following effects of MMW irradiation; analyzed on Day 1 (red bars), Day 7 (blue bars) and Day 14 (green bars). Values of cell circularity were divided into 10 subgroups and the number of cells in each group counted. The Y-axis represents the ratios of the number of irradiated cells to the number of untreated control cells for the same subgroup. Cell circularity of H1299 cells left untreated (**A**); analyzed on day 1 (Ra), day 7 (Rb) and day 14 (Rc). Cell circularity of H1299 cells irradiated under 2 min exposure regime (**B**); analyzed on day 1 (2 Ma), day 7 (2 Mb) and day 14 (2 Mc). Cell circularity of H1299 cells irradiated under the 4-min exposure regime (**C**); analyzed on day 1 (4 Ma), day 7 (4 Mb), and day 14 (4 Mc). Values of Feret’s diameter were divided into 10 subgroups and the number of cells in each group counted. Each subgroup corresponds to 25 \(\upmu\)m for all experiments.