# Morphological Changes in H1299 Human Lung Cancer Cells Following W-Band Millimeter-Wave Irradiation
## 3 Results
### MMW Irradiation Changes the Morphology and Size of H1299 Cancer Cells
Exposure for 2 min translates into 600 runs (i.e., 120 s divided by 0.2 s of a single run), providing 120 \(\upmu\)J of energy. Similarly, 4 min of exposure translates into 240 \(\upmu\)J of energy. Since \(2\times 10^{5}\) cells were irradiated in every single experiment, the estimates detailed above give an average energy dose of of 0.6 and 1.2 nJ per cell for the 2- and 4-min exposure regimes, respectively. Subsequent to the MMW exposure treatment, sham and irradiated groups were incubated under the same growth conditions during the period of observation. Cells were split appropriately several times over 14 days to maintain constant confluence rates (30%) for stringency of analyses (see methods), allowing the comparison of morphological changes between sham and treatment groups. The monitoring of the possible changes in cell morphology were examined in the control (sham) and irradiated groups (2- and 4-min exposure regimes) on Day 1 (acute response) and Day 7 and 14 following irradiation (accumulated response to irradiation, Figure 3c,d). Images of the cell morphology of sham and MMW-treated cells on Day 7 and 14 are presented in Figure 3c. Pro-apoptotic cells characterized by shrinkage and fragmentation [24] increased in the irradiated cell population as compared to the sham. Further, 10% of the population changed their form and developed protrusions 2 to 3 times longer than the cell body (Figure 3c, black arrow). The percentage of polyploids (giant cells) also increased (Figure 3c, white arrow num. 8-10), suggesting the induction of senescence [25; 26] and problems in cell division. The observed changes are represented as a ratio of the number cells in each size subgroup of the irradiated samples (2- and 4-min regimes, Figure 3d,e) as compared to the sham (Figure 3b). Under the 2-min exposure regime, samples analyzed on the same day of irradiation did not show any significant change (Figure 3d) in the population as compared to control non-irradiated cells (Figure 3b,c). However, the chronic or accumulated effects of MMW irradiation on the cell morphology were observed on Day 7 and Day 14 following exposure. The cells in the 2-min exposure regime presented a significant increase in the number of the relatively larger cells (2000-4000 \(\upmu\)m\({}^{2}\) in size) present in the H1299 cell populations. These cells, which originally constituted 25% of the sham untreated population (Figure 3b), increased in number by 1.7-2 and 6-14 times on Day 7 and Day 14, respectively (Figure 3d). In this range, the cells presented two size groups. The first group (2000-3500 \(\upmu\)m\({}^{2}\) in size) comprising of the cells in early and late stage cell division, and the second group (3500-4000 \(\upmu\)m\({}^{2}\) in size) comprising of the giant polyploid cells originally constituting only 5-15% of the untreated population (Figure 3b,c, Day 7). These results indicate that under 2 min of MMW irradiation, H1299 cells accumulate irreversible morphological changes characteristic of senescence [25; 26; 27], and/or undergo cell cycle arrest at G1/2/M phases, which is usually observed after anticancer drug treatment [28]. In contrast, the 4-min exposure regime led to the population of irradiated cells splitting into two groups of small shrunken and larger cells, respectively (Figure 3e), immediately following exposure and retained over the period of observation, as compared to the control non-irradiated cells (Figure 3c). Under the 4-min exposure regime, the majority of the population (35-40%), which were initially cells of 1000-2000 \(\upmu\)m\({}^{2}\) in size in the sham (Figure 3b), had shifted, turning into small shrunken cells (500-1500 \(\upmu\)m\({}^{2}\) in size) on the same day of irradiation (Figure 3e). Originally constituting only 15-25% of the untreated population, this group of small shrunken cells (500-1500 \(\upmu\)m\({}^{2}\) in size) increased by 1.2-2 times in number (Figure 3e).