Morphological changes in H1299 human lung cancer cells following Millimeter-wave irradiation

Efficiently targeted cancer therapy without causing detrimental side effects is necessary for alleviating patient care and improving survival rates. This paper presents observations of morphological changes in H1299 human lung cancer cells following MMW irradiation (75 – 105 GHz) at a non-thermal power density of 0.2 mW/cm2, investigated over 14 days of subsequent physiological incubation following exposure. Microscopic analyses of physical parameters measured indicate MMW irradiation induces significant morphological changes characteristic of apoptosis and senescence. The Immediate short-term stress responses translate into long-term effects, retained over the duration of the experiment(s); reminiscent of the phenomenon of Accelerated Cellular Senescence (ACS) achieving terminal tumorigenic cell growth. Further, results were observed to be treatment-specific in energy (dose) dependent manner and were achieved without the use of chemotherapeutic agents, ionizing radiation or thermal ablation employed in conventional methods; thereby overcome associated side effects. Adaptation of the experimental parameters of this study in clinical oncology concomitant with current developmental trends of non-invasive medical endoscopy alleviates MMW therapy as an effective treatment procedure for human non-small cell lung cancer (NSLC).


Introduction
Lung cancer is the leading cause of cancer deaths among men and second among women worldwide [1]. The 5-year survival rates are very low, ranging from 15.6% in the USA to as low as 8.9% in Europe, China and developing countries [1]. Lung cancer is histologically categorized as small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC). Arising from epithelial cells [2] NSCLC accounts for 80-85% of all cases. Common types of treatment methods include surgery, radiotherapy and chemotherapy [2]. However, most conventional treatments to control tumor growth are often reported to give rise to many other detrimental side effects [3,4] due to cross reactions of chemotherapy drugs with healthy tissue and use of ionizing radiations in radiotherapy. Further, post-treatment supportive care after chemotherapy is reported to go only so far as to improve survival rates very slightly [5]. Therefore, development of new and innovative therapies allowing efficient targeting of tumour growth without giving rise to unfavourable after effects is necessary to improve survival rates and alleviate patient care.
Millimeter waves (MMW) classified as non-ionizing radiation are electromagnetic fields (EMF) of extremely high frequencies  GHz) with corresponding wavelengths of 10 -1 mm. With relatively low photon energy of 0.0004 eV (1 eV = 1.6  19 J), MMW are unable to destroy inter-atomic bonds [6]; but capable of exciting rotational, torsional and longitudinal vibration modes of molecules, resulting in heating. Guidelines of the International Commission on Non-Ionizing Radiation Protection (ICNIRP) stipulates measurement of power density (PD) using units of W/m 2 for exposure of biological tissue to MMW irradiation [7]. The ICNIRP recommends limiting power density within 200 W/m 2 in order to limit adverse thermal effects on biological tissue. MMW irradiation up to a maximum power density of 5 mW/cm 2 have been shown to cause biophysical and biological effects without promoting genotoxicity and adversely increasing cell temperature (non-thermal effects) [8].
MMW irradiation leads to both activation and/or inhibition of cell growth [9 -11], changes of organelle structures and cell membrane permeability [6,12,13], alterations of DNA, RNA and proteins; and the activation or inhibition of signal transduction mechanisms [14 -16]. Such observed effects have led to a growing interest in applying non-thermal effects of MMW irradiation to target and destroy cancer cells. Previous work from our group reported non-thermal effects of MMW irradiation on human lung cancer cells [17]. In this study, we characterized the associated morphological changes using physical parameters of cell dimensions in order to better understand the mechanism of the radiation effect on the cell and thus optimize MMW irradiation therapy to treat human non-small cell lung cancer (NSLC). For this purpose, H1299 human lung cancer cells were exposed to MMW irradiation in the range of 75 -105 GHz within a stipulated non-thermal range of 2 W/m 2 . Irradiation was performed under 2 minutes and 4 minutes exposure regimes in order to determine the energy dependence of the observed effects. After irradiation, cells were incubated under physiological conditions and their physical dimensions/parameters analyzed over a period of 14 days to identify immediate and prolonged effects. The dielectric properties of the cell carrier vessels (Petri dishes) and the nutrition medium (RPMI 1640) were characterized prior to irradiation of the cell samples.

Cell culture
Human lung cancer cells, H1299 (also known as NCI-H1299 or CRL-5803) were generously provided by Professor Uri Alon of the Weizmann Institute (Rehovot, Israel). Cells were cultured in RPMI 1640 medium (Biological Industries, Beth Ha'emek, Israel) supplemented with 10% fetal bovine serum (FBS), 1% penicillin -streptomycin (Sigma, St Louis, MO, USA), and 2mM glutamine (Biological Industries, Beth Ha'emek, Israel). Cells were incubated at 37 °C with 5% CO2 supply. The proliferation rate of all cells were similar with a doubling time of 12 to 14 hours, under these conditions.

Irradiation setup
An experimental setup similar to the one described by Homenko et al. [18] was used, modified for operation in the full W-band (75-110 GHz). It consisted of a Scanning 8360B Series Swept Signal Generator and an 8757D Scalar Network Analyzer (Agilent). The sweeping frequency synthesizer was operated at 10-20 GHz serving as an input into a solid-state multiplier. A multiplying factor of 6 generated an output signal of 75-110 GHz corresponding to wavelengths (λ) of 4 -2.725 mm. The waves transmit through the reflectometer connected to two directional couplers arranged facing each other in opposite directions. The setup was calibrated for the reflection mode and the reflection coefficient measured on the analyzer. MMW were partly reflected, partly absorbed and partly transmitted through the sample. The reflection coefficient was estimated in decibels (dB) as a logarithmic ratio: , where Pr and P0 are the reflected and incident powers, respectively. Cell irradiation was performed in a sweeping regime from 75 to 110 GHz over 2,000 steps in frequency. One run over this range took 200 ms.

Penetration of MMW through Petri dish and cell growth medium
Responses of five empty Petri dishes from different manufacturers to low-intensity MMW irradiation was recorded and compared. Petri dishes from Nunc (Thermo Fisher Scientific  ) showed the best results in terms of uniformity and good transparency, hence all experiments were performed using these Petri dishes. An empty dish was placed over the lower antenna and the reflected and transmitted signals were measured in the sweeping regime from 75 to 110 GHz. Then, using a micropipette with an accuracy of 1 µ L several doses of 0.25 mL RPMI 1640 medium were successively added to obtain volumes of 0.5, 0.75, 1.00, 1.25, 1.50, 1.75 and 2.00 mL. These volumes correspond to an RPMI 1640 medium layer thickness d = 0.52, 0.80, 1.03, 1.30, 1.56, 1.82 and 2.08 mm in the petri dish. For each new volume, a new sweeping run was executed. From the obtained measurements in dB presented by equation (1) the normalized reflected and transmitted powers were calculated by (2) where P is either the reflected or transmitted power and P0 the incident power respectively.

Exposure conditions
A pyramidal horn antenna, model SGH-10 (Millitech Inc.) with aperture dimensions of 20 mm x 25 mm was used to irradiate the test samples. 1 mW incident power was used for all irradiation experiments, translating to a power density of 2 W/m 2 (0.2 mW/cm 2 ). Every single experiment involved irradiation of approximately 2x10 5 cells per dish when the cells reached 30% confluence. To investigate MMW effects on morphology, cells were irradiated under specific time regimes. Each experiment lasted up to 14 days. On day 1 cells were irradiated with MMW for 2 minutes (Group a) or 4 minutes (Group b) respectively. Following irradiation, cells were incubated at 37 ºC with 5% CO2 for up to 14 days. Slides of cell samples were prepared at specific time points -Day 1 (90 minutes after irradiation), Day 7 and Day 14. Digital images of live cells were taken using a light microscope on Day 1, Day 7 and Day 14 respectively. In order to rule out morphological changes due to cell proliferation and were not specific to MMW irradiation, two controls were used: 1) cells that underwent the same procedures but were not irradiated and 2) cells that were left untouched in the incubator. Control samples were assessed at the same time points as the irradiated cells (i.e., at Day 1, Day 7 and Day 14 of the experiment).

Microscopy and image processing
MMW effects on the morphology of the cells were examined using a Nikon fluorescent microscope (Nikon Instruments Inc., Melville, NY) at a magnification of 200x. Images were captured using a digital color-chilled 3CCD camera (Hamamatsu, Bridgewater, NJ) and visualized using the NIS Elements microscope software program (Nikon Instruments Inc.). Image processing and analysis were performed using ImageJ (Java-based image processing program developed at the National Institutes of Health, MD). Parameters of cell area, cell circularity and Feret's diameter were evaluated for irradiated and control samples. 8000 to 10000 cells were examined in each experiment. Every experiment was repeated four times.

Statistical analysis
Statistical analyses was performed using the GraphPad Prism program (GraphPad Software, La Jolla, CA). Multivariate analysis of variance (MANOVA) and Tukey-Kamer multiple comparison test were used for comparing the irradiated cells with the control groups; and for comparing the changes in irradiated cells at different exposure times. The means of the measured parameters (area, circularity and Feret's diameter) for the respective groups were considered equal for the null-hypothesis in all cases. A P-value <0.05 was considered significant for rejecting the null-hypothesis for all experiments.

Millimeter waves can penetrate through Petri dishes
In order to characterize their dielectric properties Petri dishes were irradiated from above ( Figure  1a). MMW reflected from the petri dishes were detected. The range of reflected power for frequencies 75 -100 GHz was found to be with a reduction of -15 dB to -35 dB with respect to the incident power (corresponding to 3 -0.03 % of incident power) respectively ( Figure 1b). The frequency spectrum of the reflected signal of all tested petri dishes coincided.

Millimeter waves transmit through RPMI 1640 cell growth medium without generating thermal heat
The wavelength of MMW is comparable to the layer thickness of the RPMI 1640 medium (2-3 mm) used. Additionally, MMW undergo multiple reflections in the RPMI 1640 medium. Reflected waves (from the sample) are a combination (interference) of waves reflected from the surface of the nutrition media, the bottom of the dish, and from the cells under investigation. Single (discrete) frequency irradiation regime requires fine adjustments of the distance between sample and antenna in order to obtain conditions of constructive interference. In contrast, irradiation under a continuous sweeping of frequency allows for repeated wavelength changes over each run providing conditions of constructive and destructive interference successively. This is advantageous and overcomes the necessity for fine adjustments. Thus, at least during half the duration of a single run efficient power delivery is obtained. Therefore, all experiments were conducted in the sweeping regime.
Although several studies have evaluated the real and simulated dielectric constant of RPMI medium for frequencies up to 72 GHz [19,20], such data is lacking in the MMW W-band range used in our experiments (75 -105 GHz). Hence, we tested the amount of energy that gets reflected from and penetrates through RPMI 1640 cell growth medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin. This basic medium consists of about 40 nutrition ingredients including various types of proteins present in FBS, diluted in water. The dependence of the reflected and the transmitted signals (normalized to the antenna emitting power P 0  1 mW) on the thickness of RPMI 1640 medium layer thickness in the Petri dishes was studied for different frequencies (Figure 2a). The ratio of the transmitted and incident power decreases as the thickness of the media increases. For a medium layer thickness of 2 mm, the ratio decreases by a factor of 10 -4 −10 -5 . In absolute values, this ratio corresponds to 10-100 nW of power reaching the population of cells adhered at the bottom of the dish. In other words, maximum incident power is absorbed in an RPMI 1640 medium layer thickness of 2 mm. As the medium consists mostly of water, we used the heat capacity of water to estimate the temperature increase during irradiation using (3) where is the exposure time, the heat capacity (4.18 J/g·K) and the sample mass (1.50 g). This estimation is based on the assumption that the penetrating power decreases exponentially with depth due to increased absorption of incident energy. Our calculations predict a temperature rise of no more than 0.2 -0.4 K for 2 -4 minutes of irradiation of samples immersed in 2 mL of RPMI 1640 medium. Further, temperature of the medium during irradiation was measured to be in the range of 24 -26 °C. In order to maximize power delivery to cells under irradiation and minimize attenuation loss arising from absorption by culture media, we examined the full frequency spectra (75 -105 GHZ) of the transmitted signals penetrating through petri dishes with different RPMI 1640 layer thicknesses (Figure 2b). -7.6 dB is the free space path loss (FSPL) for the 25 mm separation distance between the two +24 dB gain antennas of our setup, as detailed above (ref. Figure 1). Insertion of an empty dish increased the loss of the initial power by approximately 70% due to attenuation by the polystyrene of the dish (Figure 2b). The transmitted power level in the empty dish decreased by one order of magnitude with introduction of culture medium, corresponding to an attenuation of about -10 dB. The results indicate an RPMI layer thickness of 0.5 mm as the best working range, translating to a volume of 0.5 ml of RPMI 1640 medium. Maximum transmitted power is allowed to reach the suspended cells upon irradiation at 75 -105 GHz in this range. Therefore, all subsequent experiments were performed using this volume of culture media. To determine the dependence of observed morphological changes on the energy delivered by MMW exposure, cells were irradiated under two separate exposure regimes of 2 minutes and 4 minutes 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  1 µ W at the bottom of the petri dish (ref. Figure 2).  Morphological changes of H1299 cells following MMW irradiation (C). Prior to irradiation majority of the cells presented polygonal forms (white arrowhead 2-6) and dividing cells constituted 3-5% of the cell population (white arrow 7). Analysis of cell areas 7 days after MMW irradiation revealed 1-3% of pro-apoptotic cells (white arrow 1). 1% of cells showed characteristics of huge (giant) polyploid cells (white arrow 8 and 10). 10% of the cells showed an oval cell morphology with highly elongated protrusions, two to three times longer than the cell itself (black arrow). Bars correspond to 20 µ m. Changes in H1299 cell size irradiated under 2 minutes exposure regime (D); analyzed on day 1 (2Ma), day 7 (2Mb) and day 14 (2Mc). Cell sizes increased (3000 -4000 µ m 2 ) significantly following this treatment regime over a long-term period of 7 and 14 days. Changes in H1299 cell size irradiated under 4 minutes exposure regime (E); analyzed on day 1 (4Ma), day 7 (4Mb) and day 14 (4Mc). Cell size both decreased (0 -500 µ m 2 ) and increased (3000 -4000 µ m 2 ) significantly following this treatment regime immediately (on Day 1) and over a long-term period of 7 and 14 days respectively. 8000 -10000 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).

MMW irradiation changes morphology and size of H1299 cancer cells
The results indicate that duration of MMW exposure directly determines the extent to which cell morphology is affected. A short 2 minutes exposure does not produce any immediate effects but induces development of enlarged cells, indicative of senescence [28,29]. In contrast, a 4 minutes exposure regime providing a higher dosage of MMW energy results in both short and long-term effects.
Immediately, following exposure in this regime cells are induced to a far greater degree of shrinkage as compared to that under the 2 minutes regime; indicating acute apoptosis [27]. Further, the 4 minutes regime also induces development of enlarged polyploid cells indicative of senescence [28,29], over a long-term period of 14 days following irradiation.

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 indicated 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 a least circular shape (value 0) to a perfect circle (value 1) is used to denote the degree of dimensional roundness of an object. Increased cell circularity is a hallmark of apoptosis resulting from cytoplasmic shrinkage and cell fragmentation [30]. Majority (80%) of untreated control cells presented circularity values between 0.5 -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 minutes irradiation regime led to a population shift towards a more circular shape (0.8 -0.9) on day 1 ( Figure  4b). 7 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 already PCD cells which lost their normal form and size. Interestingly, the split in population was retained even after 14 days following irradiation. The 4 minutes irradiation regime resulted in a similar but more marked trend of population shift ( Figure 4c). As these effects persisted over day 7 to 14 day of 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 [31]. Larger FD corresponds to longer cellular extensions (i.e. protrusions); and enlarged structurally aberrant cells characteristic of senescence [32]. FD of non-irradiated cells did not change over the experimental time course (Figure 4d). The 2 minutes 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 minutes irradiation regime resulted in an FD increase over 14 days and not 7 days (Figure 4f). 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, adherent cells of the control groups changed to oval-shaped cell bodies with long protrusions following MMW exposure treatment.

Discussion
Cancer characterizes by an excessive and uncontrolled division of abnormal, malignant cells that display morphological, proliferative, and functional heterogeneity (26). Cell size is an important morphological criterion characterizing the physiological status of a cell. Majority of animal cells are 10-20 μm in diameter and rarely vary more than two folds outside of this range, suggesting that the mechanism for cell size regulation is highly conserved [33]. Changes in H1299 cell morphology following exposure to 75-110 GHz MMW for 2 and 4 minutes irradiation regimes respectively, were examined in this study. Past investigations of MMW irradiation effects on normal and cancer cells had reported distinct non-thermal biological effects using discrete narrow range(s) of frequency with very low energy [12 -13, 19, 34 -35]. MMW irradiation of lung cancer cells in the present study led to significant visible morphological changes in cell area, cell circularity and Feret's diameter. These changes were observed on the same day of exposure treatment (short-term effects), as well as over 7 -14 days post-irradiation (long-term effects). The short-term effects are likely due to regular stress responses following irradiation. However, 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 [36]. This suggests that the dramatic increment of cell size observed under the 2 minutes irradiation regime indicates a population shift towards a higher number of senescent cells induced by MMW irradiation. In contrast, the results from the 4 minutes exposure regime, which delivered a higher dose of MMW energy, indicated induction of apoptosis as well as senescence. These are favorable effects for clinical applications of MMW therapy to control tumor metastasis.
Reports of heterogeneity [26] as well as our results, demonstrate cancer cells maintain generally constant sizes throughout their lifetime. And being highly dynamic can either grow or shrink in size in response to specific conditions via robust and adaptable control mechanisms. Illustratively, alterations of cell morphologies in response to the chemical cancer drug paclitaxel have been observed with similar results [37]. Cancer cell tumorigenicity is associated with cell softening and decrease in cell stiffness arising from cytoskeletal restructuring [38]. Integral membrane proteins mediate this transition of normal cells into cancer cells [39]. Changes in cell shape and size following MMW irradiation in the present study were specific and irreversible, directly corresponding to changes of cell circularity and Feret's diameter. MMW irradiation affects cell growth [9 -11] by changing organelle structure and cell membrane permeability [6, 12 -13]. Such interactions lead to the activation and inhibition of signal transduction mechanisms due to MMW interacting with DNA, RNA and Proteins [14 -16]. Increased membrane stiffness resulting in apoptosis of cells has been shown to correspond to changes in prostate cancer cells responding to anti-neoplastic treatment [40]. These suggest the Accelerated Cellular Senescence (ACS) effect [41] occurring in an energy (dose) dependent manner observed in this study arises from the non-thermal low power density MMW exposure affecting H1299 membrane fluidity.

Conclusions
The experimental results of this study suggest MMW irradiation in the frequency range of 75 -110 GHz (W-band) promote specific morphological changes in H1299 human lung cancer cells in an energy (dose) dependent manner. Effects observed and quantified using physical dimensions/parameters of cell size, circularity and Feret's diameter demonstrate characteristic features of induced apoptosis and senescence following MMW exposure. The phenomenon of Accelerated Cellular Senescence (ACS) [41] wherein cancer cells undergo terminal growth arrest is conventionally achieved by using radiotherapy in conjunction with specific chemotherapeutic agents for targeted blockage of cellular pathways. In vivo studies suggest MMW can be used to activate Natural Killer (NK) cells aiding to reduce tumor metastasis [42]. The present study reports apoptosis and senescence of cancer cells without the use of chemotherapeutic agents, ionizing radiation or thermal ablation, thereby overcoming associated side effects [Salvo et al., 2010;Ryan, 2012]. In conjunction with the development of endoscopic methods, MMW irradiation parameters described in this study holds promising potential for the development of non-invasive procedures to treat lung cancer in the future.