Background and Aims

To dissect the immune system’s contribution to the anti-tumor activity upon FLASH and CONV radiotherapy (CONV-RT), we used different tumor models (murine lung adenocarcinoma SV2 and SV2-OVA; head & neck mEERL95; Glioblastoma GL261) transplanted in immunocompetent C57BL6/J mice, adaptive immunodeficient Swiss nude mice, and fully immunodeficient NRG (NOD-Rag1^nullIL2rg^null) mice.

Methods

Tumor cells were subcutaneously or orthotopically implanted (n=5-14) and irradiated with the eRT6/Oriatron Linac (PMB-Alcen, Fr). Subcutaneous tumors were irradiated with a single 20Gy or hypo-fractionated 3x8Gy and 2x6Gy doses using FLASH (>2,000Gy/s) or CONV-RT (~0.1-0.2Gy/s). Tumor growth delay was evaluated against unirradiated tumor bearing control mice. Orthotopic SV2-OVA lung tumors were treated with 2x6Gy, tumor growth was followed by μCT imaging and survival was monitored. Immunoprofiling was performed 3, 10 and 21 days post-RT by flow cytometry and immunofluorescence.

Results

Single and hypo-fractionated FLASH and CONV-RT were isoeffective in delaying the growth of subcutaneous tumors regardless of whether the host was immunocompetent, partially or fully immunodeficient. Interestingly, in immunocompetent mice bearing GL261 tumors, 20Gy FLASH and CONV-RT induced a complete response. Re-challenge experiments are ongoing to investigate T-cell memory. In orthotopic SV2-OVA tumors, FLASH and CONV-RT caused equal tumor growth delay, improved survival by 7 days and generated a similar immune profile. Three days post-RT, all immune populations dropped by 75% and slowly recovered at 10 days. Importantly at 21 days when 100% of control animals died, the level of CD3+ T cells was higher in both irradiated groups and correlated with enhanced survival (>90%).

Conclusions

Our results show that the radiation-induced immune response is dose rate independent. This study challenges several proffered hypotheses positing that the FLASH effect is immunologically mediated and suggests that standard radio-immunological strategies will be possible in combination with FLASH-RT.

Background and Aims

We previously identified the micro-RNA (miRNA), miR-124-3p, as a neuroprotective miRNA present in the bioactive cargo of extracellular vesicles able to counteract delayed radiation-injury in the brain (1). miR-124-3p promotes M2 polarization of microglia simultaneously reducing neuroinflammation and promoting neurogenesis. The present study was undertaken to investigate whether miR-124-3p could also be involved in neuroprotection following FLASH-RT.

Methods

Tumor-free female pediatric mice (n = 5 per group; 3 weeks old, at time of weaning) were irradiated in a single-dose regimen with 8 Gy of 6 MeV electrons administered at ultra-high dose rates (UHDR, 1 pulse, 100 Hz, 1.8 µs pulse width) or at conventional dose rates (CONV, ~ 0.1-0.2 Gy/s) with the Oriatron/eRT6 (PMB-Alcen, FR). Following comprehensive behavioral testing at 2- and 4-months post-IR (reported previously, (2)), brains were sampled, micro-dissected and flash-frozen in liquid nitrogen. Total RNA was extracted, and miR-124-3p levels were evaluated using the TaqMan Advanced miRNA Assay (ThermoFisher).

Results

Results showed that bulk brain expression of regenerative, anti-inflammatory miR-124-3p is high and at near-control levels at late time points (5 months) post-FLASH-RT, whereas miR-124-3p levels were found to be decreased significantly after CONV-RT.

Conclusions

We are in the process of analyzing more acute timepoints (< 1 mo post-irradiation) and in adult mice to investigate whether preservation of miR-124-3p levels could be used as an early marker of the sparing efficacy of FLASH-RT.

Acknowledgement: The study is funded by SNF Synergia grant (FNS CRS II5_186369)

1. R. J. Leavitt, M. M. Acharya, J. E. Baulch, C. L. Limoli, Extracellular Vesicle–Derived miR-124 Resolves Radiation-Induced Brain Injury. Cancer Research 80, 4266–4277 (2020).

2. Y. Alaghband, et al., Neuroprotection of Radiosensitive Juvenile Mice by Ultra-High Dose Rate FLASH Irradiation. Cancers (Basel) 12 (2020).

Background and Aims

Ultra-high dose rate (FLASH) radiation therapy (RT) spares damage to normal tissue compared to conventional treatment while maintaining tumor cure. However, the reported magnitude of normal tissue sparing induced by FLASH RT varies greatly between studies. The reason for this is unknown but could be due to the physical beam parameters or the radiation type itself. In this study, we aim to elucidate the sparing ability of FLASH RT across two separate beam types: electron and proton.

Methods

Eight-week-old female C57BL/6 mice were randomized to receive either electron or proton treatment. Abdominal irradiation was performed for both radiation types using either conventional (CONV, 0.16-0.5 Gy/s) or FLASH (200-300 Gy/s) dose rates to a dose ranging from 11 to 17 Gy. The 9MeV electron beam delivered a whole abdominal irradiation with a 4cm field, while the spread-out Bragg peak proton beam delivered a slightly smaller abdominal field at 2 cm due to machine limitations. Mice were subjected to crypt analysis and Kaplan-Meier analysis for survival. Animal that survived to 6 months post irradiation were examined by full necropsy.

Results

Both proton and electron FLASH demonstrated normal tissue sparing compared to CONV controls. Mice treated with electron FLASH exhibited greater recovery from weight loss compared to the CONV cohort. With proton FLASH, body weight loss was more variable but FLASH treated mice had reduced weight loss and enhanced recovery. These changes were evaluated on both beams with little variation even though there were different field sizes between them.

Conclusions

The magnitude of GI tract sparing was more pronounced following electron compared to proton FLASH irradiation, despite utilizing a larger field than the proton experiments. These data provide the first direct comparison of the FLASH effect in the GI tract between protons and electron beams and may serve as a benchmark for future studies.

Background and Aims

Using the Mobetron electron-beam linear accelerator, this study compared tumor control between ultra-high dose rate (FLASH) and conventional dose rate (CONV) electron irradiations in-vivo. We investigated the potential benefits of FLASH radiation to treat subcutaneous murine melanoma compared with CONV radiation.

Methods

Our investigation was conducted on 8-10 weeks old C57BL/6J female mice. Two melanoma cell lines, B16F10 and YUMM1.7, were subcutaneously injected into the right flank and received a single pulse of 9 MeV FLASH or an equivalent dose of CONV radiation. The flank tumors were placed centrally within the irradiation field defined by a customized 2x2cm Delrin^® collimator. Mice were stratified into three groups: 1) control/sham radiation; 2) CONV (7.8 or 8.8 Gy at 0.2 Gy/s); and 3) FLASH (7.8 or 8.8 Gy at >2E6 Gy/s). Mice were monitored 2-3 times a week for tumor growth and bodyweight changes. When the early removal criteria were met, tumors were collected for histological and cytogenetic analyses.

Results

The in-vivo experiments showed a significantly decreased tumor volume after the mice received either CONV or FLASH radiation (Student T-test, p<0.05). Tumor growth kinetics in-vivo were similar for CONV and FLASH radiation for both cell lines. Similarly, H&E staining revealed that tumors from control mice had most extensive necrosis, ranging from 30 to 90%, compared to the irradiated tumors. The CONV and FLASH irradiated mice had multifocal tumor cell characteristics such as cytomegaly and karyomegaly that may be secondary to the irradiation. We also used the multicolor fluorescence in-situ hybridization (M-FISH) technique to assess radiation-induced chromosome aberrations. The average frequencies of radiation-induced chromosomal abnormalities were comparable in the FLASH and CONV radiation-treated tumors compared to sham radiated ones.

Conclusions

The preliminary results indicated that ultra-high dose rate irradiation provided equivalent local tumor control for skin cancers in-vivo compared to conventional dose rates.