Infrared microspectroscopy to elucidate the underlying biomolecular mechanisms of FLASH radiotherapy.

BACKGROUND: FLASH-radiotherapy (FLASH-RT) is an emerging modality that uses ultra-high dose rates of radiation to enable curative doses to the tumor while preserving normal tissue. The biological studies showed the potential of FLASH-RT to revolutionize radiotherapy cancer treatments. However, the complex biological basis of FLASH-RT is not fully known yet. AIM: Within this context, our aim is to get deeper insights into the biomolecular mechanisms underlying FLASH-RT through Fourier Transform Infrared Microspectroscopy (FTIRM). METHODS: C57Bl/6J female mice were whole brain irradiated at 10 Gy with the eRT6-Oriatron system. 10 Gy FLASH-RT was delivered in 1 pulse of 1.8µs and conventional irradiations at 0.1 Gy/s. Brains were sampled and prepared for analysis 24 h post-RT. FTIRM was performed at the MIRAS beamline of ALBA Synchrotron. Infrared raster scanning maps of the whole mice brain sections were collected for each sample condition. Hyperspectral imaging and Principal Component Analysis (PCA) were performed in several regions of the brain. RESULTS: PCA results evidenced a clear separation between conventional and FLASH irradiations in the 1800-950 cm-1 region, with a significant overlap between FLASH and Control groups. An analysis of the loading plots revealed that most of the variance accounting for the separation between groups was associated to modifications in the protein backbone (Amide I). This protein degradation and/or conformational rearrangement was concomitant with nucleic acid fragmentation/condensation. Cluster separation between FLASH and conventional groups was also present in the 3000-2800 cm-1 region, being correlated with changes in the methylene and methyl group concentrations and in the lipid chain length. Specific vibrational features were detected as a function of the brain region. CONCLUSION: This work provided new insights into the biomolecular effects involved in FLASH-RT through FTIRM. Our results showed that beyond nucleic acid investigations, one should take into account other dose-rate responsive molecules such as proteins, as they might be key to understand FLASH effect.

Investigating the biochemical response of proton minibeam radiation therapy by means of synchrotron-based infrared microspectroscopy.

The biology underlying proton minibeam radiation therapy (pMBRT) is not fully understood. Here we aim to elucidate the biological effects of pMBRT using Fourier Transform Infrared Microspectroscopy (FTIRM). In vitro (CTX-TNA2 astrocytes and F98 glioma rat cell lines) and in vivo (healthy and F98-bearing Fischer rats) irradiations were conducted, with conventional proton radiotherapy and pMBRT. FTIRM measurements were performed at ALBA Synchrotron, and multivariate data analysis methods were employed to assess spectral differences between irradiation configurations and doses. For astrocytes, the spectral regions related to proteins and nucleic acids were highly affected by conventional irradiations and the high-dose regions of pMBRT, suggesting important modifications on these biomolecules. For glioma, pMBRT had a great effect on the nucleic acids and carbohydrates. In animals, conventional radiotherapy had a remarkable impact on the proteins and nucleic acids of healthy rats; analysis of tumour regions in glioma-bearing rats suggested major nucleic acid modifications due to pMBRT.