# 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  
H1299 lung cancer cells were irradiated with MMWs and were subsequently incubated under the physiological conditions before further evaluation. Analyses of the irradiated and control cells were conducted on Day1 (90 min post irradiation), Day 7, and Day 14 (Figure 3a). Exposure power density was maintained at 0.2 mW/cm\({}^{2}\) over the duration of exposure, much below the maximum permissible exposure of 1 mW/cm\({}^{2}\), as stipulated by the US Federal Communications Commission [19; 20; 21; 22]. This power density along with the estimates and measurements of the temperature increase mentioned above allowed for the thermal effects arising from this irradiation regime to be ruled out. Therefore,  
Figure 2: Dependence of the reflected and the transmitted signals on the level of the RPMI 1640 medium layer’s thickness (**A**). Signals were evaluated for different frequencies ranging from 75 to 100 GHz. For the empty dish, only the transmitted signal and not the reflected signal is presented. Shown is one representative sample of seven dishes that were irradiated for each frequency. Exposure times were a few seconds and the temperature of the medium was measured to be 24–26 \({}^{\circ}\)C. Dependence of the transmitted power through the Petri dish on the frequency of irradiation (**B**). The signals were normalized to the antenna emitting power P\({}_{0}\approx 1\) mW on an empty dish. the effects observed experimentally were considered non-thermal in nature. Prior to irradiation, cell morphology was examined by microscopy. H1299 cells were observed to be adherent flattened cells with a thickness of less than 5 \(\upmu\)m, indicating that the measurements of the cell area are representative of their actual size. Figure 3: Pictogram of the experimental strategy (**A**), depicting the time schedule and analytical conditions (R—Non-irradiated control retained in the incubator, 2M—two minutes, 4M—four minutes. IR—irradiation, SR—Control cells that underwent the same procedures as the irradiated cells without being irradiated, IMAGE—Microscopy and image analysis). Cells were irradiated for two minutes (2M) or four minutes (4M). Image analysis was performed on day 1 (short-term, a), day 7 (long-term, b), or day 14 (long-term, c). Controls were assessed at the same time points as the irradiated cells (i.e., Ra, day 1; Rb, day 7; and Rc, Day 14 of the experiment). Quantification of the size distribution of control non-irradiated cell areas (**B**) for day 1 (Ra, red line), day 7 (Rb, green line), and day 14 (Rc, violet line). Cell areas were grouped into 10 intervals (500 \(\upmu\)m\({}^{2}\) each) and the number of cells in each group counted and examined by microscopic observation followed by image analysis of cell sizes using the ImageJ program. The observations demonstrate non-significant changes amongst the sham (non-irradiated) cell size subgroups examined over the experimental period (14 days). Morphological changes of H1299 cells following MMW irradiation (**C**). Prior to irradiation, the majority (35-45%) of the cells presented polygonal-form cells in the interphase stage with areas in the range of 1000-2000 \(\upmu\)m\({}^{2}\) (white arrowhead 2-6), with 3-15% of small shrunken/pro-apoptotic cells in the range of 500-1000 \(\upmu\)m\({}^{2}\), and 12-15% of dividing cells in the range 2000-3500 \(\upmu\)m\({}^{2}\) (white arrow 7) constituting the cell population. Analysis of cell areas 7 and 14 days after MMW irradiation revealed an increased number of pro-apoptotic cells (white arrow 1). In total, 1-3% of cells showed characteristics of huge (giant) polyploid cells (white arrow 8 and 10) in the range of 3500-5000 \(\upmu\)m\({}^{2}\); 10% of the cells showed an oval cell morphology with highly elongated protrusions, two to three times longer than the cell itself (black arrow).