# 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
Bars correspond to 20 \(\upmu\)m. Changes in H1299 cell size irradiated under the 2-min exposure regime (**D**); analyzed on day 1 (2 Ma), day 7 (2 Mb), and day 14 (2 Mc). Cells ranging 2000-4000 \(\upmu\)m\({}^{2}\) in size increased significantly by 2-14 times following this treatment regime over a long-term period of 7 and 14 days, comprising of the cells in different cell division stages and the giant polyploid cells. Changes in H1299 cell size irradiated under the 4-min exposure regime (**E**); analyzed on day 1 (4 Ma), day 7 (4 Mb), and day 14 (4 Mc). Following the 4-min exposure regime, the irradiated cells divided into two populations: One group consisting of shrunken cells (500-1500 \(\upmu\)m\({}^{2}\) in size) undergoing apoptosis and a second group consisting of large senescent and mitotically arrested giant enlarged cells (2500-3500 \(\upmu\)m\({}^{2}\) in size). Cells groups ranging 500-1500 \(\upmu\)m\({}^{2}\) and 2500-3500 \(\upmu\)m\({}^{2}\) in size increased significantly by 2-5 and 1.6-1.9 times, respectively, following this treatment regime immediately over a long-term period of 7 and 14 days, respectively. The morphological changes of H1299 cells the under the 4-min exposure regime are observed to be stable and accumulated over the period of observation (14 days). In total, 8000-10,000 cells were analyzed for every single experiment using Image J. Error bars represent the standard deviations of four biologically independent experiments (N = 4); \(p\)-values \(<\) 0.05 was considered statistically significant with \({}^{*}\) representing \(p\)\(<\) 0.05 (for a versus c or b versus c) and \(\#\) representing \(p\)\(<\) 0.05 (for a versus b). Prior to quantifying the changes in their morphology following irradiation, cells were divided into 10 subgroups of 500 \(\upmu\)m\({}^{2}\) each based on their area size (Figure 3b) as a non-irradiated reference. The number of cells for each subgroup from the controls (non-irradiated and incubator control; see methods) was determined by counting during imaging observation in the light microscope. The square root of the observed top-view area was found to be 25-30 \(\upmu\)m for the average population. Non-irradiated control cells were observed to be in interphase with polygonal, slightly elongated, or oval forms. The majority of these cells (35-45%) in the interphase stage presented areas in the range of 1000-2000 \(\upmu\)m\({}^{2}\) (Figure 3b). In total, 3-15% of the cells were relatively smaller (500-1000 \(\upmu\)m\({}^{2}\)), accounting for pro-apoptotic cells. Dividing cells with areas ranging from 2000-2500 \(\upmu\)m\({}^{2}\) and 3000-3500 \(\upmu\)m\({}^{2}\) constituted about 12-15% of the population (Figure 3b). Huge (giant) polyploid cells, about 2.5-3.5 times larger than the main cell population with areas ranging from 3500 \(\upmu\)m\({}^{2}\) to 5000 \(\upmu\)m\({}^{2}\), constituted about 1-3% of the cell population (Figure 3b). The observations demonstrate non-significant changes amongst all the sham (non-irradiated and incubator control; see methods) cell size subgroups examined over the experimental period (14 days) (Figure 3b). The results showed that H1299 cell populations present a heterogeneous morphology corresponding to the physiological status of the cells in their cell cycle states, concomitant with studies reporting cancer cell diversity and genetic instability [23]. To determine the dependence of the observed morphological changes on the energy delivered by MMW exposure, cells were irradiated under two separate exposure regimes of 2 and 4 min, respectively. Depending on the frequency, a single run at a maximum incident power of 1 mW incident power was almost completely attenuated in 2 mL of RPMI 1640 medium, reaching a level of about P \(\approx\) 1 \(\upmu\)W at the bottom of the Petri dish (Figure 2). In this regime, the energy delivered to the bottom of the dish was 200 nJ (1 \(\upmu\)W \(\times\) 200 ms).