Nanoscale investigation into the cellular response of glioblastoma

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Nanoscale investigation into the cellular response of glioblastoma cells exposed to protons Ewelina Lipiec, Bayden R. Wood, Andrzej Kulik, Wojciech M. Kwiatek, and Giovanni Dietler Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b01497 • Publication Date (Web): 25 May 2018 Downloaded from http://pubs.acs.org on May 25, 2018

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Analytical Chemistry

Nanoscale Investigation into the Cellular Response of Glioblastoma Cells Exposed to Protons

Ewelina Lipiec1,2,3*, Bayden R. Wood3, Andrzej Kulik2, Wojciech M. Kwiatek1, Giovanni Dietler2 1

Institute of Nuclear Physics, Polish Academy of Sciences, PL-31342 Krakow, Poland,

2

Institute of Physics, Laboratory of Physics of Living Matter, Ecole Polytechnique Fédérale de

Lausanne (EPFL), CH-1015 Lausanne, Switzerland, 3

Centre for Biospectroscopy and School of Chemistry, Monash University, 3800, Victoria,

Australia *[email protected] Abstract Exposure to ionising radiation can induce cellular defence mechanisms including cell activation, rapid proliferation prior to metastasis and in extreme cases can result in cell death. Herewith we apply infrared nano- and micro-spectroscopy combined with multidimensional data analysis to characterise the effect of ionising radiation on single glioblastoma nuclei isolated from cells treated with 10 Gy of X-rays or 1 Gy and 10 Gy of protons. We observed chromatin fragmentation related to the formation of apoptotic bodies following X-ray exposure. Following proton irradiation we detected evidence of a DNA conformational change (B-DNA to A-DNA transition) related to DNA repair and accompanied by an increase in protein content related to the synthesis of peptide enzymes involved in DNA repair. We also show that proton exposure can increase cholesterol and sterol ester synthesis, which are important lipids involved

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in the metastatic process changing the fluidity of the cellular membrane in preparation for rapid proliferation.

Introduction Hadron therapy is beneficial for the treatment of many kinds of tumours, including the central nervous system, head and neck, eye, lung, sarcomas, prostate, and many paediatric cancers.1 However, this type of cancer treatment is rarely applied compared to other techniques including X-rays, gamma rays or electron irradiation. Hadron therapy requires advanced instrumentation including cyclotrons to accelerate protons. Despite the poor availability of cyclotron facilities there is a lack of research investigating the influence of protons on living matter. These two factors have slowed down the implementation of Hadron therapy as therapeutic tool. The effect of irradiation should be studied holistically and include studies on single molecular, cellular organelles, single cells, tissues, single organisms and at the population level. Research on radiation dose–dependent biological effects (such as DNA damage: single and double strand breaks cross–links, oxidative damage and conformational change) enhances our understanding of the mechanisms leading to cell death.2, 3 Current practice is to use biochemical assays to detect damage to cellular components. However, the application of the biochemical methods may affect the biological samples leading to changes in their structure due to non–physiological chemical substances and complex preparation procedures.4-6 Moreover, most of biochemical methods are not intended to study the effect of radiation at the single cell/single cellular nucleus level. Therefore, there exists a need for complementary techniques to confirm cellular damage arising from radiation exposure. The main purpose of this study was to apply a state-of-the-art AFM-IR spectroscopic system to investigate the spectral changes that occur in single cellular nuclei isolated from cells treated with protons and X-rays. Classical infrared spectroscopic based techniques are well known for their uniqueness as non-invasive tools for the identification


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of vibrational structure in biological materials and offer a novel way to examine radiation damage at the single cell level.4-6 The radiation damage of nucleic acids,

7, 8

peroxidation in

model phospholipid and membrane systems 9 and structural changes in protein 10, 11 have been previously studied by molecular spectroscopic approaches. However, none of those studies were performed with nanometer spatial resolution. In classical infrared microspectroscopy, submicrometer resolution is not attainable in the IR range because this technique is diffraction limited and hence not applicable to intracellular mapping. In a previous study performed by our group we proved that nuclei isolation is an ideal technique to study the response of radiation using SR–FTIR (Synhrotron Radiation – Fourier Transform Infrared spectroscopy).4 The spectral profiles of isolated nuclei and cells are similar, however, in nuclei spectra the DNA bands are more clearly defined4. Additionally, cellular nuclei have been found to be 100 times more sensitive to radiation exposure than cytoplasm.12 Here we report, for the first time, the application of infrared nano-spectroscopy to detect changes induced by radiation damage and the cellular response in single nuclei. Infrared nano-spectroscopy has previously been applied to studies of various biological systems such as such single mammalian cells 13-15 bacteria 16, 17 and amyloid fibrils.18, 19 Dazzi et al.

16

reported anticancer drug detection in single cells, by

localization of rhenium-carbonyl complex inside cells after a 1h-incubation at 10 µL of organometallic conjugate. Kennedy et al

13

applied recently AFM-IR in mammalian cells

studies. Researchers focus on the biochemical heterogeneity of cells. They have monitored the influence of the material absorption coefficient as well as the influence of thickness of the sample located under the tip on the observed cantilever deflection while scanning complex biological systems. This research proved that local heterogeneity of complex systems could be studied by image analysis with the Minkowski function.13 More recently Perez-Guaita et al. applied multispectral AFM-IR to distinguish malaria parasite organelles in fixed red blood cells.20



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Experimental Section Proton irradiation Brain tumor cells (A-172 giloblastoma) suspended in cell culture medim (DMEM + 10 % of Fetal Bovine Serum) were irradiated with 60 MeV protons from the Proteus C -235 cyclotron at the Institute of Nuclear Physics PAN in Krakow, Poland. Two dosages of protons were applied:1 Gy and 10 Gy. The deadly dose of X-rays -10 Gy was applied as a negative control for comparison. The X-rays were obtained from 250 kV X-ray tube at the Institute of Nuclear Physics PAN in Krakow, Poland. After appropriate incubation time (24 h, 48h) cellular nuclei were extracted according the procedure reported by Junaid et al.

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Trypsinized cells were,

centrifuged (1400 r.p.m) for 4 mins and then suspended in ice–cold nuclear extraction buffer [320 mM sucrose, 5 mM MgCl2, 10 mM HEPES and 1% Triton X–100 (pH 7.4)]. Then, the solution was vortexed gently for 10 s and incubated on ice for 10 mins. In order to collect nuclei the suspension was centrifuged at 2000 r. p. m. Nuclei were washed two times in freshly prepared buffer [320 mM sucrose, 5 mM MgCl2 and 10 mM HEPES (pH 7.4)]. Freshly isolated nuclei were suspended in saline solution and then deposited on a ZnSe prisms or CaF2 windows for the measurements.

Infrared nano-spectroscopy and mapping Single spectra of dried control and irradiated nuclei were collected at a spectral resolution of 4 cm–1 in a spectral range of 3600 cm-1 – 1000 cm-1 with 1024 cantilever ringdowns coadded at each

wavenumber position. NanoIR infrared spectrometer (from Anansys Instruments Santa


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Barbara, California) equipped in tunable nanosecond optical parameter oscillator (OPO) IR laser was applied for measurements (EPFL Lausanne, Switzerland). The scan areas were not bigger that 15 μm per 15 μm (pixel size 50 nm x 50 nm or smaller). The scan rate was set to 0.01-0.02 Hz. To improve the statistics all measurements were repeated 3 times independently. From each group of cells (control, exposed to particular type of radiation (1 Gy and 10 Gy protons, 10 Gy X-rays) and incubated for 24h or 48 hours) 2-4 cellular nuclei were mapped. AFM and Infrared maps were analysed using the SPIP software. Images were flattened (global correction, polynominal fit 2nd order).

Infrared micro-spectroscopy A droplet of nuclei immersed in saline solution was deposited between two CaF2 windows. Infrared spectra of single cellular nuclei fully immersed in saline solution were collected at the infrared microspectroscopy beamline at the Australian Synchrotron using a Bruker V80v FTIR and Hyperion microscope, with narrow band MCT detector. An aperture of 10 µm x 10 µm was used. Spectra were collected in transmission mode in the spectral range of 4000 cm–1 – 750 cm– 1

with a spectral resolution of 4 cm–1. For each spectrum 64 interferograms were co-added.

Single spectra analysis Each spectrum was smoothed using Savitzky-Golay algorithm (2-nd order of polynominal, 3 smoothing points). The cellular spectra were calculated by subtracting the saline solution spectrum from the cell + saline spectrum using an appropriate scaling factor and manually adjusting each spectrum to minimize the residual area between the cell spectra and saline solution spectra in the 2500-1850 cm-1 spectral region 22. Then Principal Component Analysis were applied using Unscrambler (CAMO) software.4-6 Several PCA models were tested in two spectral ranges: fingerprint (1750 cm-1 – 1030 cm-1 ) and CH2, CH3 stretching modes (3000 cm-



1

– 2800 cm-1). Three most significant PCs were taken into consideration PC-1, PC-2 and PC-

3.

Results and Discussion In the current study the distribution of nucleic acids, proteins and lipids were mapped in nuclei extracted from control cells and those exposed to radiation. Typical results obtained for nuclei isolated 48 hours post irradiation are presented in Figure 1. One can analyse the distribution of the absorption at 1230 cm-1 corresponding to the O-P-O asymmetric stretching motions from the DNA backbone, 1660 cm-1 assigned to the Amide I band and 2952 cm-1 from asymmetric stretching of methyl groups from lipid chains. The corresponding AFM topography images and the distribution of AFM cantilever frequency is also presented in Figure 1. The morphology of control and irradiated nuclei are very different. Control nuclei are more homogenous than their irradiated counterparts. Inside the nuclei there is just one smooth spherical area of concentrated chromatin that is clearly visible, which is the nucleolus. We have observed that the rough granular distribution of nucleic acids, proteins and lipids was typical for the nuclei isolated from a cell exposed to 10 Gy of X-rays. The observed nuclei fragmentation pattern is typical of processes that occur in cells exposed to serious damaging factors such as radiation and is a signature of programmed cell death – apoptosis. 23 This is a cellular defence mechanism, which prevents inflammation. Not all proteins located inside cells are recognizable by the immunological system, therefore to prevent the inflammation, cells form apoptotic bodies, which are composed of condensed cellular debris enclosed in the cellular membrane.23 We did not observe any type of granular apoptotic bodies forming following proton irradiation. However, we detected crystallized material on the surface of cell nuclei. The stiffness of this material was much higher than the stiffness of the nuclear membrane, which is clearly visible in cantilever frequency distribution plot (Fig. 1). The distribution of these stiff structures


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corresponds to the distribution of methyl groups from lipid chains (absorbtion at 2952 cm-1). Therefore, AFM-IR proved, that these crystals contain mainly lipids. The intermediate changes after 24 hours of incubation post irradiation are shown in the Supporting Information (section S1). The results confirm nuclear fragmentation following 10 Gy of X-rays exposure and the appearance of crystalized lipid deposits on the surface of nuclei isolated from cells exposed to 10 Gy of protons. However, significantly less lipid crystal deposits were detected after 24 hours of incubation compared to 48 hours.

Figure 1 AFM-IR maps of nucleic acids (1230 cm-1), proteins (1660 cm-1) and lipids (2952 cm1 ) together with AFM topographies and cantilever frequency distribution in single cellular nuclei isolated from control cells and cells treated with 1 Gy and 10 Gy of 60 Mev protons and 10 Gy of X-rays.



In order to identify the type of lipid, single AFM-IR spectra were collected from the regions containing the lipid structures and other regions devoid of them. Representative spectra are presented in Figure 2. Spectra collected from lipid crystal deposits (Fig 2 a) clearly indicate a high contribution from lipids in the nuclear material. To help identify the lipid we subtracted spectra collected from the neighbouring places free of the lipid crystals. A typical spectrum is presented in Figure 2 c, which shows strong absorption at 2920 cm-1 and at 1464 cm-1 assigned to CH2 stretching and CH2 scissoring deformations, respectively and are indicative of cholesterol.24-27 The cholesteryl ester infrared makers were also found in spectra collected from the lipid crystals, including the C=O bond of the ester linkage at 1728 cm-1, CH2 scissoring deformations at 1464 cm-1 and CH stretching at 2848 cm-1).25-27 Additionally, the presence of the C=C stretching band at 1650 cm-1 25, 26 is indicative of unsaturated fatty acids. We did not take into our consideration band at 1620 cm-1 because this wavenumber is located on the boundary between two laser stages and such peak could be artificial. Sterols, mainly cholesterol and cholesteryl-esters modulate the fluidity of the lipid bilayer and are thus important in the function of the cellular membrane.27 Therefore, the presence of sterol and sterol esters may be attributed to cellular preparation prior to rapid proliferation associated with metasasis. According to the literature a number of inhibitors of fatty acid synthesis play important roles in metastases prevention.27


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Figure 2 The spectra of lipids accumulated in cells treated by radiation: a) the AFM topography, b) AFM-IR map of absorption at 2956 cm-1 (lipid distribution) in cellular nuclei isolated from cell 48 hours after treatment with 10 Gy of 60 MeV protons, c) AFM-IR spectra collected from the place where lipid crystal were observed (1) and from an area free of lipid crystals (2), the spectral difference (1-2) indicates the presence of cholesterol and sterol esters.

A full description of the molecular changes occurring in nucleic acids, proteins and lipids following radiation exposure can be obtained by careful analysis of the spectra. Here we are proposing a comparison of nano and microscale infrared spectra. Spectra collected at the nanoscale are very sensitive to localalised structural changes 4 and are therefore the spectra are highly variable due to complex cell structure. However, averaged nanoscale spectra (Fig 3a)



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should resemble averaged microscale spectra, like in our case SR-FTIR spectra collected from single cellular nuclei (Fig 3 b). A comparison between averaged AFM-IR and averaged SRFTIR spectra are presented in Figure 3. The area underneath the Amide I band varies significantly between the spectra recorded using the different modalities because of the contribution of H-O-H bending mode at ~1640 cm-1 from water

22

. SR-FTIR spectra were

collected from wet nuclei (fully immersed in saline solution) while AFM-IR spectra were recorded from dried nuclei. The Amide I band is relatively stronger (in comparison with other bands) in AFM-IR spectra compared to the SR-FTIR spectra because the saline solution is subtracted from the saline + cell SR-FTIR spectrum. For the AFM-IR spectra this subtraction was not performed and hence this region is excluded from the analysis and modelling. Therefore, the spectral differences in the area of the Amide I mode were not taken into consideration when looking for spectral markers indicative of radiation influence. This region was also excluded from the statistical analysis of the SR-FTIR data. Strong absorption at, 1418 cm-1 and 1468 cm-1 and in the 2750 cm-1 – 3100 cm-1 spectral range in SR-FTIR as well as AFM-IR spectra indicate that irradiation with 10 Gy of protons strongly increases the lipid content. SR-FTIR also confirmed the presence of mainly cholesterol and cholesteryl esters. We have detected a number of spectral changes related to DNA damage and repair. An intensity decrease in the base stacking mode at 1713 cm-1 is caused by base–pair damage including purine, pyrimidine dimer formation and 6–4 lesions

4-6

was observed in SR-FTIR spectra of

nuclei isolated from cells treated by 1 Gy, 10 Gy of protons and 10 Gy of X rays. In AFM-IR spectra this band was not clearly resolved from the C=O stretching band from lipids at 1742 cm-1. Another significant infrared marker of radiation damage is a shift in the O–P–O asymmetric stretching band to lower wavenumber value, indicating conformational changes occurring to the DNA, 26 which could play an important role in the resistance to DNA damage.28 This partial shift from 1220 cm-1 to 1240 cm-1 was observed in SR-FTIR spectra of nuclei


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isolated from cells treated by 10 Gy of protons after 48 hours of incubation (Fig 3 and Fig. S1). The DNA conformation is very sensitive to moisture changes.4, 28 The DNA transition from B to A upon drying has previously been reported in cells.28 However, conformational changes in response to radiation damage are not easy detectable in dried samples, as measured by AFMIR.

Figure 3 Averaged AFM-IR (a) and averaged SR-FTIR (b) spectra collected form nuclei isolated from control cells and cells incubated for 48 hours after exposure to 1 Gy and 10 Gy of 60 MeV protons and 10 Gy of 60 KeV X-rays.



An intensity decrease of O–P–O asymmetric stretching mode observed for irradiated cells, is possibly associated with the fact that after radiation exposure, the cells are stopped in the G1 phase during DNA repair. Cells in the G1 phase show lower absorbance of bands associated with phosphodiester bonds. 29 Irradiation with 10 Gy of protons resulted in an absorbance increase in the Amide II band at 1550 cm-1 in both SR-FTIR and AFM-IR spectra collected from nuclei isolated from cells incubated for 24 h and 48 h after radiation exposure. This observation is possibly related to DNA repair. The enzymes involved in DNA repair are mainly proteins and an increase in the amount of protein should cause an increase in the intensity of Amide II peak in FTIR spectra. We have compared spectra obtained from AFM-IR and SRFTIR and their derivatives (Fig 3, Supporting Information Fig. S2 – Fig. S4) and both indicated spectral changes typical for apoptosis after X-rays exposure and an induction sterols synthesis in cells irradiated with protons. In order to reduce data dimensionality but also to demonstrate variability and reproducibility within the data sets we have used Principal Component Analysis (PCA). PCA was applied to the raw AFM-IR spectra and second derivative SR-FTIR spectra, respectively because the latter technique showed significant baseline variation. For each data set (SR-FIIR and AFM-IR) two spectral ranges from 1800 cm–1 to 1030 cm–1 and 3000 cm–1 – 2750 cm–1 were analysed, separately. In the data set of SR-FTIR spectra, the Amide I spectral region was excluded from the analysis because of high contribution of H-O-H bending at ~1640 cm-1 22 as it was mentioned before. PCA was performed using the NIPALS algorithm in Unscrambler (Camo, Norway) software using leave-out-one cross validation and the data mean-centered. The results for spectra isolated 48 hours after radiation exposure are presented in Figure 4. In the spectral range from 1800 cm–1 to 1030 cm–1 four clusters of spectra are clearly visible in both Scores Plots (Fig


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4 a and Fig 4 b). The clustering of AFM-IR spectra is more evident than SR-FTIR spectra due to higher sensitivity of nano-spectroscopy for local chemical changes related to radiation exposure and also smaller and homogenous baseline contribution in AFM-IR data, which enabled us to perform the decomposition on raw spectra. However, both Loadings Plots indicated that the same chemical changes might be followed at micro and nanoscale. Along PC1 (explaining 85% and 53% percentage of total variance in SR-FTIR and AFM-FTIR data, respectively) the separation of cluster of spectra collected from nuclei isolated from cells treated by 10 Gy is clearly visible. These clusters are located on negative side of PC-1 in both Scores Plots. Both Loading Plots show a negatively correlation to Amide II band at 1548 cm-1 (SRFTIR), 1532 cm-1 (AFM-IR) indicating an increase of protein content in irradiated nuclei. It is also negatively correlated with the bands at 1720 cm-1 from the base staking mode and C=O from lipids indicating DNA damage and changes in the lipid structure. PC-2 and PC-3 loadings show that the separation along these variables is related to DNA changes. SR-FTIR spectra recorded from nuclei irradiated with 10 Gy of protons and 10 Gy of X-rays located at negative site of PC-2 and PC-3. Loadings Plots confirmed that the position of the asymmetric stretching phosphodiester peak in SR-FTIR spectra appears at 1229 cm-1 control nuclei and at and at 1219 cm-1 for cells irradiated with 1 Gy of protons. This shift is related to a B-like DNA to A-like DNA transition occurring in response to the DNA repair process. PC-2 and PC-3 loadings indicated that the spectral position of asymmetric stretching phosphodiester band is also important in clustering of the AFM-IR spectra, however, the shift is not that clearly visible in spectra of dry samples compared to SR-FTIR spectra of fully immersed in saline solution nuclei.

A separation was also observed in CH2 and CH3 stretching spectral regions (Fig. 4 c and d). Due to higher sensitivity for local changes AFM-IR spectra (Fig. 4 c) are better separated than



SR-FTIR spectra (Fig 4 d). PC-1 explains 66% of total variance within SR-FTIR and AFM-IR data sets. For each Scores plot 4 clusters corresponding the nuclei isolated from control cells and cells treated with two doses of protons and one dose of X-rays are observed. Loadings Plots indicate that the bands responsible for this separation are CH2 stretching at 2918 cm-1 and 2873 cm-1, characteristic of cholesterol and cholesetryl esters and are typical for spectra of nuclei irradiated with 1 Gy and 10 Gy of protons. The separation along PC-2 explaining 20 % clearly indicates the influence of the radiation showing all AFM-IR spectra of nuclei isolated from irradiated cells are located on the negative side of PC-2, while spectra of control nuclei are at positive site. PC-3 explains 5 % of total variance and it is dominated by peak at 2844 cm-1 (CH2 stretching). The position of the methyl band at 2844 cm-1 was detected in control cells and cells that had recovered after being irradiated with 10 Gy of protons, which were preparing themselves proliferation. PCA analysis performed on spectra of nuclei incubated for 24 hours after radiation exposure are shown in the Supporting Information (Fig. S5).


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Figure 4 PCA anlysis of AFM-IR spectra and SR-FTIR spectra: (a) Scores and Loading Plots of AFM-IR spectra in 1750 cm-1 – 1030 cm-1 spectral range, (b) Scores and Loading Plots of SR-FTIR spectra in 1750 cm-1 – 1030 cm-1 spectral range, (c) Scores and Loading Plots of AFM-IR spectra in 3000 cm-1 – 2800 cm-1 spectral range, (d) Scores and Loading Plots of SRFTIR spectra in 3000 cm-1 – 2800 cm-1 spectral range.



Conclusion In summary the spectra of nuclei isolated from the cells exposed to 10 Gy of X-rays show heterogeneity of the nucleic acids, proteins (chromatin) and lipids indicative of chromatin condensation, which is characteristic of late apoptosis. We were also able to detect lipid deposits in nuclei treated with 10 Gy of protons. We have spectroscopically proven that the newly synthesised lipids contain mainly cholesterol and cholesteryl esters. This sub-micron detail would not be possible using conventional or synchrotron infrared sources because of the low sensitivity and spatial resolution. However, SR-FTIR spectra confirmed that lipid content in nuclei isolated from cells treated by 10 Gy of protons increases dramatically. These results are in agreement with the increasing body of evidence 30 that lipid accumulation especially cholesterol and cholesteryl esters are a characteristic for aggressive cancer cells, which are involved in the production of membranes before cellular division. The lipids are a hallmark of aggressive cancer cells, producing membranes for rapid cell proliferation.30 On the other hand, lipid biosynthesis plays an important role in cancer cell migration and invasion, and also in the induction of tumor angiogenesis. These processes are considered to be crucial for the dissemination of tumour cells and for the formation of metastases, which constitute the main cause of cancer mortality.31 The outcome of this work will help to better understand the interaction between single cells and protons or X-rays, which should be taken into consideration when designing a strategy for the treatment of giloblastoma. Supporting Information Available: Figure S1 presents AFM-IR maps of nucleic acids (1230 cm-1) proteins (1660 cm-1) and lipids (2952 cm-1) together with AFM topographies and cantilever frequency distribution in single cellular nuclei isolated from control cells and cells treated by 1 Gy and 10 Gy of 60 Mev protons and 10 Gy of X-rays


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Figure S2 presents averaged SR-FTIR (A) and AFM-IR (B) spectra collected form nuclei isolated from control cells and cells incubated for 24 hours after exposure to 1 Gy and 10 Gy of 60 MeV protons and 10 Gy of 60 KeV X rays Figure S3 shows averaged second derivatives of SR-FTIR spectra collected form nuclei isolated from control cells and cells incubated for 24 hours after exposure to 1 Gy and 10 Gy of 60 MeV protons and 10 Gy of 60 KeV X rays Figure S4 shows averaged second derivatives of SR-FTIR spectra collected form nuclei isolated from control cells and cells incubated for 48 hours after exposure to 1 Gy and 10 Gy of 60 MeV protons and 10 Gy of 60 KeV X rays Figure S5 shows PCA analysis of AFM-IR spectra and SR-FTIR spectra: A) Scores and Loading Plots of AFM-IR spectra in 1750 cm-1 – 1030 cm-1 spectral range, B) Scores and Loading Plots of AFM-IR spectra in 3000 cm-1 – 2800 cm-1 spectral range, C) Scores and Loading Plots of SR-FTIR spectra in 1750 cm-1 – 1030 cm-1 spectral range, D) Scores and Loading Plots of SRFTIR spectra in 3000 cm-1 – 2800 cm-1 spectral range; A detailed description of Figure S5 is also presented. References: 1. Tommasino F, Durante M. Proton Radiobiology. Cancers 2015; 7:353–381. 2. Ugenskiene R, Lekki J, Polak W, Prise K M, Folkard M, Veselov O, Stachura Z, Kwiatek WM, Zazula M, Stachura J. Double strand breaks formation as a response to X-ray and targeted proton irradiation. J Nucl Instr and Meth in Phys Res B 2007;260:159–163. 3. Schettino G, Folkard M, Michael BD, Prise KM, Low-dose binary behavior of bystander cell killing after microbeam irradiation of a single cell with focused c(k) x rays, Radiat Res 2005;163:332–336.



4. Lipiec E, Bambery KR, Heraud P, Kwiatek WM, McNaughton D, Tobin MJ, Vogel C, Wood BR. Monitoring UVR induced damage in single cells and isolated nuclei using SR-FTIR microspectroscopy and 3D confocal Raman imaging. Analyst, 2014;139:4200–4209. 5. Lipiec E, Bambery KR, Heraud P, Hirschmugl C, Lekki J, Kwiatek WM, Tobin MJ, Vogel C, Whelan D, Wood BR. Synchrotron FTIR shows evidence of DNA damage and lipid accumulation in prostate adenocarcinoma PC-3 cells following proton irradiation. J Mol Struct 2014;1073:134–141. 6. Lipiec E, Bambery KR, Lekki J, Tobin MJ, Vogel C, Whelan DR, Wood BR, Kwiatek WM. SR-FTIR coupled with principal component analysis shows evidence for the cellular bystander effect. Radiat Res 2015;184(1):73-82,   7.

Sailer K, Viaggi S, Nusse M. Radiation-induced structural modifications in dsDNA analysed by FT-Raman spectroscopy. Int J Radiat Biol 1996;69:601–613.

8. Dovbeshko G, Gridina NY, Kruglova EB, Pashchuk O P. FTIR Spectroscopy studies of nucleic acid damage. Talnta 2000;53:233–246. 9. Sailer K, Viaggi S, Nusse M. Kinetics of Radiation- and Cytochrome c-Induced Modifications in Liposomes Analysed by FT-Raman Spectroscopy. Biochim Biophys Acta 1997;1329:259–268. 10. Synytsya A. Alexa, P. de Boer, J. Loewe, M. Moosburger, M. Wurkner, M. Volka, K. Raman spectroscopic study of serum albuminutes: an effect of proton– and gamma–irradiation, J. Raman Spectrosc. 2007;38:1646–1655. 11. Gault N, Lefaix JL. Infrared Microspectroscopic Characteristics of Radiation-Induced Apoptosis in Human Lymphocytes. Radiat. Res. 2003;160:238–250. 12. Nias AHW. An Introduction to Radiobiology, New York, Wiley, Nov 2, 1990.


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Acknowledgements This research was supported by a National Science Center of the Republic of Poland (Narodowe Centrum Nauki—NCN), within the framework of a project entitled "Chromatine role in formation of chromosomal aberrations" No. 2014/13/D/NZ1/01014. Prof. B.R.W. is supported by an Australian Research Council (ARC) Future Fellowship (FT120100926). Prof. Małgorzata Lekka is gratefully acknowledged for providing A172 and DU145 cells for the experiment. The authors would like to gratefully acknowledge also Ph.D. Agnieszka Panek for X- rays irradiation of cells, Ph.D. Eng. Jan Swakoń and the staff of the Bronowice Cyclotron Centre for proton beam irradiation.



For TOC only

The entire Table of Contents graphic is original and was created by one of the co-authors (Ewelina Lipiec), using original AFM-IR images, AFM-IR spectra. TOC was created using a Microsoft Office (Power Point) program.


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Nanoscale investigation into the cellular response of glioblastoma

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