Background and Aims

Using glioblastoma as model, the aim of the present study was to investigate the role of G2/M arrest in tumor response to FLASH-RT and to characterize Damage Associated Molecular Patterns (DAMPs) that might amplify anti-tumor immunogenic response.

Methods

In vitro, GL261, H454 and PDGC2159 GBM and HaCat normal cells were synchronized (or not) in G2/M phase using a CDK1 (9uM) or PLK1 (25nM) inhibitor 24hours before 20Gy FLASH-RT (2.10³Gy/s, 2 pulses of 10Gy, 100Hz) or CONV-RT (~0.1-0.2Gy/s, 10Hz) with eRT6 (Jorge, 2019). Calreticulin, HSPA5, ATP, HMGB1, DNA release, micronuclei formation and cGAS-STING-type I IFN response were investigated. In vivo, murine GL261 and PDGC2159 GBM cells were orthotopically grafted to C57Bl6 and Swiss nude mice. Mice were treated with a single dose of 10Gy delivered Whole-Brain either with FLASH (≥10⁷Gy/s, 1 pulse) or CONV-RT (~0.1-0.2Gy/s). Tumor control, normal brain toxicity, immune response and in situ vaccination were evaluated.

Results

In vitro, the level of micronuclei positive cells was similar after FLASH and CONV (40% vs 0% in non-RT) and HMGB1 mRNA level was enhanced (+1.8fold) in FLASH vs CONV irradiated samples. G2/M blockade significantly increased micronuclei formation (+20%), and cGAS mRNA level (+2.33fold) in FLASH vs CONV irradiated samples. Other markers were not modified. In vivo experiments are ongoing.

Conclusions

These preliminary results support a G2/M-dependent release of DAMPs after FLASH irradiation that might trigger downstream immune response. Experiments are ongoing to characterize this response along with anti-tumor efficacy and normal toxicity in immune-deficient/competent mice.

Background and Aims

Using glioblastoma as model, the aim of the present study was to investigate the role of G2/M arrest in tumor response to FLASH-RT and to characterize Damage Associated Molecular Patterns (DAMPs) that might amplify anti-tumor immunogenic response.

Methods

In vitro, GL261, H454 and PDGC2159 GBM and HaCat normal cells were synchronized (or not) in G2/M phase using a CDK1 (9uM) or PLK1 (25nM) inhibitor 24hours before 20Gy FLASH-RT (2.10³Gy/s, 2 pulses of 10Gy, 100Hz) or CONV-RT (~0.1-0.2Gy/s, 10Hz) with eRT6 (Jorge, 2019). Calreticulin, HSPA5, ATP, HMGB1, DNA release, micronuclei formation and cGAS-STING-type I IFN response were investigated. In vivo, murine GL261 and PDGC2159 GBM cells were orthotopically grafted to C57Bl6 and Swiss nude mice. Mice were treated with a single dose of 10Gy delivered Whole-Brain either with FLASH (≥10⁷Gy/s, 1 pulse) or CONV-RT (~0.1-0.2Gy/s). Tumor control, normal brain toxicity, immune response and in situ vaccination were evaluated.

Results

In vitro, the level of micronuclei positive cells was similar after FLASH and CONV (40% vs 0% in non-RT) and HMGB1 mRNA level was enhanced (+1.8fold) in FLASH vs CONV irradiated samples. G2/M blockade significantly increased micronuclei formation (+20%), and cGAS mRNA level (+2.33fold) in FLASH vs CONV irradiated samples. Other markers were not modified. In vivo experiments are ongoing.

Conclusions

These preliminary results support a G2/M-dependent release of DAMPs after FLASH irradiation that might trigger downstream immune response. Experiments are ongoing to characterize this response along with anti-tumor efficacy and normal toxicity in immune-deficient/competent mice.

Background and Aims

In this study, we investigated the effects of tumor oxygen tension on the anti-tumor efficacy of ultra-high-dose-rate (FLASH) radiotherapy (RT).

Methods

U87 glioblastoma cells were xenografted in Swiss Nude mice and irradiated using a single 20-Gy dose administered at UHDR (2 pulses, 100 Hz, 1.8 µs pulse width, 0.01 s delivery) or CONV (~ 0.1 Gy/s) dose rates with the Oriatron/eRT6 (PMB, CHUV) under normoxia, hypoxia (vascular clamp), and hyperoxia (carbogen breathing). In situ oxygen tension was measured during and following irradiation using an OxyLite probe. Tumor growth was monitored using caliper measurements and tumor were sampled for RNA and protein profiling (GIF, UNIL). Metabolic analysis and ROS measurements were performed in vitro using Seahorse XF96 Analyzer and CellROX.

Results

Surprisingly, the anti-tumor efficacy of FLASH-RT was not affected by hypoxia in this U87 xenograft model, whereas hypoxia induced radioresistance with CONV-RT. Genomic profiling revealed a decrease in hypoxia signaling in the FLASH-treated compared to the CONV-treated and control tumors 24h post-RT. Oxidative metabolism was also altered in response to FLASH-RT. Real-time tumor oxygen readout, ROS levels, and metabolic testing at different oxygen tensions and timepoints post-RT are in progress.

Conclusions

FLASH-RT anti-tumor efficacy does not seem to be affected by hypoxia supporting a differential role for oxygen signaling between FLASH and CONV-RT and opening new venues for clinical application of FLASH-RT in a subset of highly radiation resistant tumors.

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