## 4 Rationale of Proton Beam Therapy
Cells were treated with 1 and 10 Gy of protons at the Institute of Nuclear Physics PAN in Krakow (Poland) after an incubation time of 24 and 48 h cellular nuclei were extracted. A drop of nuclei in saline solution was placed between two CaF\({}_{2}\) windows. Infrared spectra of single cellular nuclei in saline solution were acquired at the infrared microspectroscopy beamline at the ANSTO Australian Synchrotron in Clayton (Australia) using a Bruker V80 FTIR and HYPERION microscope equipped with a narrow band MCT detector. An aperture of 10 \(\times\) 10 \(\upmu\)m was set. Spectra acquisition was performed in the transmission mode in the 4000\(-\)750 cm\({}^{-1}\) wavenumber range with a spectral resolution of 4 cm\({}^{-1}\). Second derivative analysis and PCA were used for analyzing the acquired spectra. The spectral features of isolated nuclei and cells were analogous, but the DNA bands were more evident in nuclei spectra. This result could be due to the higher sensitivity of cell nuclei to radiation exposure than that of cytoplasm. The authors focused their attention on the peak located at 1230 cm\({}^{-1}\) due to the O-P-O asymmetric stretching mode from the DNA, the band at 1660 cm\({}^{-1}\) attributed to the Amide I band, and the features observed at 2952 cm\({}^{-1}\) resulting from asymmetric stretching of methyl groups from lipid contribution. The analysis of SR-\(\upmu\)FTIR spectra evidenced that exposure to 10 Gy of protons causes a strong increase in the lipid content with the presence of principally cholesterol and cholesteryl esters. Spectral modifications related to DNA damage and repairing processes were also evident. The decrease in the intensity of the 1713 cm\({}^{-1}\) feature can be attributed to the base-pair damage including purine, pyrimidine dimer formation, and lesions, and it was observed in the spectra from nuclei isolated from cells treated by 1 and 10 Gy of protons. Radiation exposure also caused a shift in the O-P-O asymmetric stretching band towards a lower wavenumber value, indicating the occurrence of DNA conformational changes. This shift from 1220 to 1240 cm\({}^{-1}\) was present in the SR-\(\upmu\)FTIR spectra of nuclei exposed to 10 Gy of protons and extracted after 48 h of incubation. The decrease in the intensity of this peak can be related to the experimental evidence that after radiation exposure, the cells were stopped in the G1 phase during DNA repairing processes. Cells in the G1 phase are characterized by a smaller band related to phosphodiester bonds. Exposure to 10 Gy of protons can be also associated with an increase in the intensity of the Amide II band at 1550 cm\({}^{-1}\) in SR-\(\upmu\)FTIR spectra collected from nuclei extracted from cells 24 and 48 h after irradiation. This change can be related to the enzymes involved in DNA repair (see Figures S2-S4 in the Supplementary Materials of Reference [56], freely available, and Reference [19]) and to sterol synthesis processes in cells irradiated with protons. PCA was performed in two wavenumber regions (i.e., 1800-1030 and 3000-2750 cm\({}^{-1}\)), and the Amide I spectral range was not included in the analysis because of the presence of water contribution due to the saline solution used during the spectra acquisition. Results confirmed the lipid accumulation (mainly cholesterol and cholesteryl esters) in cancer cells. This represents a biomarker for aggressive cancer cells that produce membranes for rapid cell proliferation. In addition, lipids play an important role in cancer cell migration and invasion processes that are of relevant importance in the distribution of tumor cells and the formation of metastases [56].