Background and Aims

FLASH radiotherapy (FLASH-RT) is an emerging modality that uses high dose rates of radiation to enable curative doses to the tumour while preserving normal tissue. The impressive 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.

Within this context, our aim is to get deeper insights into the biomolecular mechanisms underlying FLASH-RT through Fourier Transform Infrared Microspectroscopy (FTRIM).

Methods

C57B6 female mice were whole brain irradiated at 10 Gy with eRT6-Oriatron (PMB-Alcen, France) as already described (Montay-Gruel, PNAS, 2019). FLASH-RT was delivered in 1 pulse of 1.8 μs and CNV at 0.1 Gy/s. Brain were sampled and prepared for analysis 24h post-RT.

FTIRM was performed at the MIRAS-BL of ALBA Synchrotron using the BRUKER 3000 Hyperion microscope coupled to a Vertex 70 spectrometer. Infrared raster scanning maps of the whole mice brain sections were collected for each sample condition with a beam spot size of 100x100 mm². Principal Component Analysis (PCA) was performed in different ROIs of the brain sections.

Results

Preliminary PCA results evidenced a clear separation between conventional and FLASH-RT irradiations in the fingerprint region. An analysis of the loading pots revealed that most of the variance accounting for the separation between groups was associated to distinct conformational changes in the Amide I band. Also, DNA rearrangements varied as a function of the irradiation configuration. Cluster separation was also present in the lipid region, being correlated with changes in the CH_x vibrational bands. Relevant biomarkers for lipid modifications (relative content of lipids, lipid chain alterations, lipid peroxidation, unsaturation) indicated distinct structural and biochemical perturbations in FLASH-RT with respect to conventional irradiations. Vibrational features were ROI-dependent.

Conclusions

This work provided new insights into the biomolecular effects involved in FLASH-RT through FTIRM.

Background and Aims

The use of ultra-high dose rate irradiation (FLASH RT) has shown promising effects compared to conventional dose rates in terms of reduced normal tissue toxicity while maintaining tumor control. The normal tissue sparing of FLASH RT may be related to differences in tissue oxygen tension during treatment. Here, we evaluate a non-invasive methodology for quantification of oxygen levels in tumors and tissue before and after irradiation.

Methods

Multi Spectral Optoacoustic Tomography (MSOT) imaging was used to perform high-resolution 3D in vivo imaging of the oxygen saturation (sO₂) throughout the body of mice, which is used as a surrogate for pO₂. The oxygen levels in vivo were manipulated by using different carrier gases in the sedation apparatus delivering isoflurane, which included carbogen, 100% oxygen, 21% oxygen (air), and 10% oxygen. The influence of sedation strategy with and without ketamine was also explored. Mice were irradiated with either conventional or FLASH dose rates to a dose of 14Gy to the abdomen, and the crypt assay was used to quantify the acute toxicity post-irradiation. The in vivo oxygen status was correlated to the treatment response.

Results

The FLASH group had a substantially greater quantity of regenerating crypts over the conventional mice across various parameters and the sparing effect of FLASH seems to diminish with increasing in vivo oxygen levels.

Conclusions

The normal tissue sparing induced through FLASH was dependent on the oxygen tension of the tissue under irradiation. By manipulating the oxygen levels in vivo, the magnitude of the FLASH effect can be even further expanded. These data lay the groundwork for future novel treatment strategies of oxygen-controlled FLASH radiation therapy.

Background and Aims

Translation of FLASH radiation for localized ocular cancers may prospectively increase vision retention rates and reduce the need for motion tracking in comparison to conventional radiotherapy techniques. We have developed a platform to enable preclinical ocular FLASH irradiation with kV x-rays and will present the results of a pilot study of FLASH normal tissue sparing effects from murine whole-eye irradiation.

Methods

Healthy 8 week old C57BL6J mice were irradiated with 150 kVp x-rays to doses up to 26 Gy at FLASH (right eye) and CONV (left eye) dose rates, respectively, using a FLASH-capable rotating anode x-ray tube and custom immobilization. Both eyes were irradiated for direct comparison of dose rate effects on the same animal. In vivo dose measurements were performed using thermoluminescent microcube dosimeters, each encased in a 3-mm diameter holder and inserted into an enucleated mouse carcass. Visual acuity was assessed using scotopic electroretinography (ERG) up to 2 months post irradiation. Histological changes were assessed through H&E staining of the harvested eyes

Results

Measured dose rates at the eye center were 52.6 ± 2.6 Gy/s and 1.1 ± 0.1 Gy/s at FLASH and CONV settings respectively. Thorough functional and histological assessment remains underway. Preliminary results of ERG response to visual stimuli show vision worsened at 26 Gy for both dose rates, yet that FLASH-treated mice higher response than CONV-treated mice at 2 months after irradiation. A corresponding difference was observed histologically, with degradation of the photoreceptor layer in CONV-treated mice.

Conclusions

Vision appears to be better-retained following FLASH x-ray irradiation, with preliminary histological confirmation. This first demonstration of FLASH normal tissue sparing effects in a mouse eye model presents a unique and promising translation opportunity for clinical FLASH treatment. Further ongoing studies will explore the long-term effects of FLASH radiation on vision and its efficacy on melanoma-type tumors.

Background and Aims

Deciphering the molecular and cellular mechanisms of the FLASH effect is key to determine the clinical applications of FLASH radiotherapy. In the lung, FLASH has been shown to limit the occurrence of severe side-effects such as pulmonary fibrosis. Although these complications develop over months after radiation injury, previous results from the lab, precision-cut lung slices, showed that FLASH irradiation spared the replication capacity of lung cells in the 24h following radiation injury. These data indicate that early responses correlate with the late decrease in lung toxicities (i.e. severe pneumonitis evolving towards pulmonary fibrosis) observed after FLASH irradiation.

Methods

In order to investigate the molecular and cellular mechanisms underlying the FLASH effect in the lung, we performed single cell RNAseq analysis at early (i.e. 24 hours) and late (i.e. 5 months) time points after whole thorax irradiation at a dose of 13 Gy delivered either in CONV or FLASH mode with the ElectronFLASH device.

Results

Preliminary analysis suggests that FLASH reduces the activation of endothelial cells (i.e proinflammatory and procoagulant state) and impacts differentially the expression of genes associated to lipid metabolism in some epithelial cell populations. These comprehensive single cell RNAseq analysis will open the way to mechanistic hypothesis explaining the FLASH sparing effect in the healthy lung tissue.

Conclusions

In summary, this study will provide key molecular and cellular events occurring specifically after FLASH compared to CONV lung irradiation. We propose to validate the most relevant results using precision-cut lung slices. In a broader context, this study will allow a better understanding of radiation-induced toxicities in the lung and will hopefully support the optimization radiotherapy treatments using FLASH.

Background and Aims

To explore the x-ray beam characteristics necessary to trigger the FLASH effect, Drosophila melanogaster larvae were exposed to ultrahigh dose-rate (UHDR) or conventional radiotherapy dose-rate (CONV-RT) x-rays from one of two sources: 10 MV x-rays at the TRIUMF ARIEL beamline and 120 kVp x-rays delivered with a conventional x-ray tube.

Methods

At TRIUMF, Drosophila larvae were irradiated with 10 MV x-rays at UHDR (72-102 Gy/s) or CONV-RT dose-rates (0.04-0.06 Gy/s) with doses between 9.4 and 16.8 Gy. Larvae were immobilized within acrylic phantoms with tape at 5 and 13mm depths in solid water. Larvae that survived to adulthood 8 days post irradiation were tracked for an additional 8 days.

With the x-ray tube, larvae were irradiated with 120 kVp x-rays collimated with a rotating tungsten shutter. UHDR dose-rates of 114 Gy/s were achieved by delivering single exposures at 25 mA. CONV-RT irradiations were performed by delivering 5 fractions at 5 mA with 25 seconds between exposures. Both UHDR and CONV-RT groups of 10 larvae were irradiated with exposure settings of 25 to 100 ms (~5-20 Gy). Survival to adulthood was assessed at 6 days post irradiation by counting emerged flies.

Results

While all surviving flies irradiated with 10 MV x-rays to 9.4 Gy remained alive 8 days later, those irradiated with higher doses exhibited diverging survival between FLASH and CONV-RT groups. When exposed to ~16.5 Gy, the survival decreased to 78% and 25% for the FLASH and CONV-RT groups, respectively. A difference in survival was also observed for larvae irradiated with 120 kVp x-rays. The 11 Gy FLASH group showed 40% greater survival than the 9.8 Gy CONV-RT group.

Conclusions

The FLASH effect was observed for total body irradiations of Drosophila melanogaster delivered with clinically relevant high-energy 10 MV x-rays as well as low-energy 120 kVp x-rays suitable for small animal irradiations.