Jonathan Ollivier (Switzerland)
CHUV OncologyAuthor Of 6 Presentations
ZEBRAFISH EMBRYOS: A HIGHTHROUPUT MODEL TO CHARACTERIZE BEAM PARAMETERS ABLE TO TRIGGER THE FLASH EFFECT
IMPACT OF DOSE RATE DELIVERED WITH ELECTRON, PROTON AND PHOTON BEAMS ON THE DEVELOPMENT OF ZEBRAFISH EMBRYOS
A PURSUIT FOR A HIGH-THROUGHPUT INDICATOR OF THE FLASH EFFECT
Abstract
Background and Aims
Despite an immense research interest in FLASH-RT, the precise beam requirements for obtaining the FLASH effect have still not been elucidated. Yet, there is an increasing number of reports assigning the name FLASH to beams and irradiators without any supportive biological data.Methods
Currently, the FLASH effect can be confirmed only in vivo, which requires time-consuming animal studies and corresponding ethical approvals. The FLASH community would therefore greatly benefit from a high-throughput FLASH beam indicator that can validate an UHDR beam for FLASH-RT. Such indicator has to generate an observable that follows the same dependency on temporal beam characteristics as the FLASH effect.Results
We used our published data on sparing of the normal mouse brain and killing of GBM to monitor the impact of gradually changing electron beam parameters (dose rate, dose per pulse) on the occurrence of the FLASH effect. These data were considered as a template to investigate responses of various high-throughput assays over the same range of beam parameters. In particular, we studied assays that previously showed differential response to our FLASH and Conv beams: H2O2 yield, O2 depletion, plasmid DSB, lipid peroxidation and zebrafish embryo. Only the length of zebrafishes grown from irradiated embryos showed the dependency on beam parameters mimicking the cognitive protection in vivo with low energy electron beam (Oriatron).Conclusions
Since mechanistic differences between different types of beams (protons, X-ray) are possible, further investigations are mandatory to confirm universal validity of this model as a general FLASH indicator.CHARACTERIZATION OF DAMAGE ASSOCIATED MOLECULAR PATTERNS AFTER FLASH RADIOTHERAPY TO AMPLIFY ANTI-TUMOR IMMUNE RESPONSE
Abstract
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.
PLASMID DNA DAMAGES AFTER FLASH VS CONVENTIONAL DOSE RATE IRRADIATIONS IN VARIOUS OXYGEN CONDITIONS
Abstract
Background and Aims
In our work we thought to compare the effects of conventional (CONV) vs ultra-high dose rate (UHDR) by quantifying DNA strand breaks (SB) after irradiation of plasmid-DNA (pBR322) under various oxygen concentrations.
Methods
Supercoiled pBR322 was irradiated dry or in water using a 6 MeV FLASH-validated electrons beam, with increasing doses (1-100 Gy) and dose per pulse (0.01 Gy/s (CONV), 5.0*102 to 5.6*106 Gy/s (UHDR)) and at atmospheric (21%), physoxic (4%) and hypoxic (0.5%) oxygen level. The increase of relaxed (R) and linear (L) plasmid forms after irradiation was quantified by agarose gel electrophoresis and used to compute single and double SB yields.
Results
Dry, atmospheric conditions cause similar yields of SB in CONV and UHDR. Aqueous conditions shows higher SB yields as expected. Physoxia induces radioprotection compare to atmospheric condition: 50% of R at 10 Gy (4% O2) vs 2 Gy (21% O2), but no difference relative to dose rate. Hypoxia revealed higher SB yields than physoxia in CONV (50% of R at 6 Gy) but 2x less SB in UHDR for doses >30 Gy (see figure for L).
Conclusions
First results in dry condition suggest that direct effects are not involved in FLASH. In aqueous condition, 4% oxygen mimicking healthy tissues shows no difference between UHDR and CONV, while 0.5% oxygen mimicking tumors shows less damages in UHDR. These results are opposite to the preclinical results showing the FLASH effect. Thus, plasmid irradiation might be useful to understand DNA damage at UHDR but seems barely relevant to investigate the FLASH effect at the biological level.
NOT JUST HEALTHY TISSUE SPARING: HYPOXIA DOES NOT IMPACT FLASH-RT ANTI-TUMOR EFFICACY
Abstract
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)