Oxygen Monitoring in Model Solutions and In Vivo in Mice During Proton Irradiation at Conventional and FLASH Dose Rates.

FLASH is a high-dose-rate form of radiation therapy that has the reported ability, compared with conventional dose rates, to spare normal tissues while being equipotent in tumor control, thereby increasing the therapeutic ratio. The mechanism underlying this normal tissue sparing effect is currently unknown, however one possibility is radiochemical oxygen depletion (ROD) during dose delivery in tissue at FLASH dose rates. In order to investigate this possibility, we used the phosphorescence quenching method to measure oxygen partial pressure before, during and after proton radiation delivery in model solutions and in normal muscle and sarcoma tumors in mice, at both conventional (Conv) (∼0.5 Gy/s) and FLASH (∼100 Gy/s) dose rates. Radiation dosimetry was determined by Advanced Markus Chamber and EBT-XL film. For solutions contained in sealed glass vials, phosphorescent probe Oxyphor PtG4 (1 µM) was dissolved in a buffer (10 mM HEPES) containing glycerol (1 M), glucose (5 mM) and glutathione (5 mM), designed to mimic the reducing and free radical-scavenging nature of the intracellular environment. In vivo oxygen measurements were performed 24 h after injection of PtG4 into the interstitial space of either normal thigh muscle or subcutaneous sarcoma tumors in mice. The "g-value" for ROD is reported in mmHg/Gy, which represents a slight modification of the more standard chemical definition (µM/Gy). In solutions, proton irradiation at conventional dose rates resulted in a g-value for ROD of up to 0.55 mmHg/Gy, consistent with earlier studies using X or gamma rays. At FLASH dose rates, the g-value for ROD was ∼25% lower, 0.37 mmHg/Gy. pO2 levels were stable after each dose delivery. For normal muscle in vivo, oxygen depletion during irradiation was counterbalanced by resupply from the vasculature. This process was fast enough to maintain tissue pO2 virtually unchanged at Conv dose rates. However, during FLASH irradiation there was a stepwise decrease in pO2 (g-value ∼0.28 mmHg/Gy), followed by a rebound to the initial level after ∼8 s. The g-values were smaller and recovery times longer in tumor tissue when compared to muscle and may be related to the lower initial endogenous pO2 levels in the former. Considering that the FLASH effect is seen in vivo even at doses as low as 10 Gy, it is difficult to reconcile the amount of protection seen by oxygen depletion alone. However, the phosphorescence probe in our experiments was confined to the extracellular space, and it remains possible that intracellular oxygen depletion was greater than observed herein. In cell-mimicking solutions the oxygen depletion g-vales were indeed significantly higher than observed in vivo.

Improving the efficiency of small animal 3D-printed compensator IMRT with beamlet intensity total variation regularization.

PURPOSE: There is growing interest in the use of modern 3D printing technology to implement intensity-modulated radiation therapy (IMRT) on the preclinical scale that is analogous to clinical IMRT. However, current 3D-printed IMRT methods suffer from complex modulation patterns leading to long delivery times, excess filament usage, and less accurate compensator fabrication. In this work, we have developed a total variation regularization (TVR) approach to address these issues. METHODS: TVR-IMRT was used to optimize the beamlet intensity map, which was then converted to a thickness of the corresponding compensator attenuation region in copper-doped polylactic acid (PLA) filament. IMRT and TVR-IMRT heart and lung plans were generated for two different mice using three, five, or seven gantry angles. The total compensator thickness, total variation of compensator beamlet thicknesses, total variation of beamlet intensities, and exposure time were compared. The individual field doses and composite dose were delivered to film for one plan and gamma analysis was performed. RESULTS: In total, 12 mice heart and lung plans were generated for both IMRT and TVR-IMRT cases. Across all cases, it was found that TVR-IMRT reduced the total variation of compensator beamlet thicknesses and beamlet intensities by 54 ± 4 % $54\pm 4\%$ and 50 ± 3 % $50\pm 3\%$ on average when compared to standard 3D-printed compensator IMRT. On average, the total mass of compensator material consumed and radiation beam-on time were reduced by 45 ± 6 % $45\pm 6\%$ and 24 ± 4 % $24\pm 4\%$ , respectively, whereas dose metrics remained comparable. Heart plan compensators were printed and delivered to film and subsequent gamma analysis performed for each of the single fields as well as the composite dose. For the composite delivery, a passing rate of 89.1% for IMRT and 95.4% for TVR-IMRT was achieved for a 3 % / 0.3 $3\%/0.3$ mm criterion. CONCLUSIONS: TVR can be applied to small animal IMRT beamlet intensities to produce fluence maps and subsequent 3D-printed compensator patterns with significantly less complexity while still maintaining similar dose conformity to traditional IMRT. This can simplify/accelerate the 3D printing process, reduce the amount of filament required, and reduce overall beam-on time to deliver a plan.

Ultrafast Tracking of Oxygen Dynamics During Proton FLASH.

PURPOSE: Radiation therapy delivered at ultrafast dose rates, known as FLASH RT, has been shown to provide a therapeutic advantage compared with conventional radiation therapy by selectively protecting normal tissues. Radiochemical depletion of oxygen has been proposed to underpin the FLASH effect; however, experimental validation of this hypothesis has been lacking, in part owing to the inability to measure oxygenation at rates compatible with FLASH. METHODS AND MATERIALS: We present a new variant of the phosphorescence quenching method for tracking oxygen dynamics with rates reaching up to ∼3.3 kHz. Using soluble Oxyphor probes we were able to resolve, both in vitro and in vivo, oxygen dynamics during the time of delivery of proton FLASH. RESULTS: In vitro in solutions containing bovine serum albumin the O2 depletion g values (moles/L of O2 depleted per radiation dose, eg, µM/Gy) are higher for conventional irradiation (by ∼13% at 75 µM [O2]) than for FLASH, and in the low-oxygen region (<25 µM [O2]) they decrease with oxygen concentration. In vivo, depletion of oxygen by a single FLASH is insufficient to achieve severe hypoxia in initially well-oxygenated tissue, and the g values measured appear to correlate with baseline oxygen levels. CONCLUSIONS: The developed method should be instrumental in radiobiological studies, such as studies aimed at unraveling the mechanism of the FLASH effect. The FLASH effect could in part originate from the difference in the oxygen dependencies of the oxygen consumption g values for conventional versus FLASH RT.

Stoichiometric analysis of protein complexes by cell fusion and single molecule imaging.

The composition, stoichiometry and interactions of supramolecular protein complexes are a critical determinant of biological function. Several techniques have been developed to study molecular interactions and quantify subunit stoichiometry at the single molecule level. However, these typically require artificially low expression levels or detergent isolation to achieve the low fluorophore concentrations required for single molecule imaging, both of which may bias native subunit interactions. Here we present an alternative approach where protein complexes are assembled at physiological concentrations and subsequently diluted in situ for single-molecule level observations while preserving them in a near-native cellular environment. We show that coupling this dilution strategy with fluorescence correlation spectroscopy permits quantitative assessment of cytoplasmic oligomerization, while stepwise photobleaching and single molecule colocalization may be used to study the subunit stoichiometry of membrane receptors. Single protein recovery after dilution (SPReAD) is a simple and versatile means of extending the concentration range of single molecule measurements into the cellular regime while minimizing potential artifacts and perturbations of protein complex stoichiometry.