Oxygen supplementation in anesthesia can block FLASH effect and anti-tumor immunity in conventional proton therapy.

BACKGROUND: Radiation-induced neurocognitive dysfunction is a major adverse effect of brain radiation therapy and has specific relevance in pediatric oncology, where serious cognitive deficits have been reported in survivors of pediatric brain tumors. Moreover, many pediatric patients receive proton therapy under general anesthesia or sedation to guarantee precise ballistics with a high oxygen content for safety. The present study addresses the relevant question of the potential effect of supplemental oxygen administered during anesthesia on normal tissue toxicity and investigates the anti-tumor immune response generated following conventional and FLASH proton therapy. METHODS: Rats (Fischer 344) were cranially irradiated with a single high dose of proton therapy (15 Gy or 25 Gy) using FLASH dose rate proton irradiation (257 ± 2 Gy/s) or conventional dose rate proton irradiation (4 ± 0.02 Gy/s), and the toxicities in the normal tissue were examined by histological, cytometric and behavioral analysis. Glioblastoma-bearing rats were irradiated in the same manner and tumor-infiltrating leukocytes were quantified by flow cytometry. RESULTS: Our findings indicate that supplemental oxygen has an adverse impact on both functional and anatomical evaluations of normal brain following conventional and FLASH proton therapy. In addition, oxygen supplementation in anesthesia is particularly detrimental for anti-tumor immune response by preventing a strong immune cell infiltration into tumoral tissues following conventional proton therapy. CONCLUSIONS: These results demonstrate the need to further optimize anesthesia protocols used in radiotherapy with the goal of preserving normal tissues and achieving tumor control, specifically in combination with immunotherapy agents.

Proton therapy is a type of precise radiotherapy that can have reduced side effects. Children undergoing proton therapy are often given a general anesthetic, supplemented with high oxygen levels as a measure of safety. However, the consequences of modifying the oxygen concentration in the treatment have not been studied. In this study, we evaluated the consequences of adding oxygen in the anesthesia in a model of brain tumor after conventional proton therapy and a new radiotherapy technique, FLASH proton therapy. We observed that oxygen supplementation can cause more brain damage in FLASH proton therapy and block anti-tumor immune cell infiltration into the tumor in conventional proton therapy. Overall, this study should be taken into consideration when designing new protocols of radiotherapy, specifically those including FLASH proton therapy and combinations with immune-targeted treatments.

New calibration setup for quantitative DCE-US imaging protocol: Toward standardization.

BACKGROUND: The DCE-US (Dynamic Contrast-Enhanced Ultrasonography) imaging protocol predicts the vascular modifications compared with Response Evaluation Criteria in Solid Tumors (RECIST) based mainly on morphological changes. A quantitative biomarker has been validated through the DCE-US multi-centric study for early monitoring of the efficiency of anti-angiogenic cancer treatments. In this context, the question of transposing the use of this biomarker to other types of ultrasound scanners, probes and settings has arisen to maintain the follow-up of patients under anti-angiogenic treatments. As a consequence, radiologists encounter standardization issues between the different generations of ultrasound scanners to perform quantitative imaging protocols. PURPOSE: The aim of this study was to develop a new calibration setup to transpose the DCE-US imaging protocol to the new generation of ultrasound scanners using both abdominal and linear probes. METHODS: This calibration method has been designed to be easily reproducible and optimized, reducing the time required and cost incurred. It is based on an original set-up that includes using a concentration splitter to measure the variation of the harmonic signal intensity, obtained from the Area Under the time-intensity Curve (AUC) as a function of various contrast-agent concentrations. The splitter provided four different concentrations simultaneously ranging from 12.5% to 100% of the initial concentration of the SonoVue contrast agent (Bracco Imaging S.p.A., Milan, Italy), therefore, measuring four AUCs in a single injection. The plot of the AUC as a function of the four contrast agent concentrations represents the intensity variation of the harmonic signal: the slope being the calibration parameter. The standardization through this method implied that both generations of ultrasound scanners had to have the same slopes to be considered as calibrated. This method was tested on two ultrasound scanners from the same manufacturer (Aplio500, Aplioi900, Canon Medical Systems, Tokyo, Japan). The Aplio500 used the settings defined by the initial multicenter DCE-US study. The Mechanical Index (MI) and the Color Gain (CG) of the Aplioi900 have been adjusted to match those of the Aplio500. The reliability of the new setup was evaluated in terms of measurement repeatability, and reproducibility with the agreement between the measurements obtained once the two ultrasound scanners were calibrated. RESULTS: The new setup provided excellent repeatability measurements with a value of 96.8%. Once the two ultrasound scanners have been calibrated for both types of probes, the reproducibility was excellent with the agreement between their respective quantitative measurement was at the lowest 95.4% and at the best 98.8%. The settings of the Aplioi900 (Canon Medical Systems) were adjusted to match those of the Aplio500 (Canon Medical Systems) and these validated settings were for the abdominal probe: MI = 0.13 and CG = 34 dB; and for the linear probe: MI = 0.10 and CG = 38 dB. CONCLUSION: This new calibration setup provided reliable measurements and enabled the rapid transfer and the use of the DCE-US imaging protocol on new ultrasound scanners, thus permitting a continuation of the therapeutic evaluation of patients through quantitative imaging.

Proton FLASH Radiation Therapy and Immune Infiltration: Evaluation in an Orthotopic Glioma Rat Model.

PURPOSE: FLASH radiation therapy (FLASH-RT) is a promising radiation technique that uses ultrahigh doses of radiation to increase the therapeutic window of the treatment. FLASH-RT has been observed to provide normal tissue sparing at high dose rates and similar tumor control compared with conventional RT, yet the biological processes governing these radiobiological effects are still unknown. In this study, we sought to investigate the potential immune response generated by FLASH-RT in a high dose of proton therapy in an orthotopic glioma rat model. METHODS AND MATERIALS: We cranially irradiated rats with a single high dose (25 Gy) using FLASH dose rate proton irradiation (257 ± 2 Gy/s) or conventional dose rate proton irradiation (4 ± 0.02 Gy/s). We first assessed the protective FLASH effect that resulted in our setup through behavioral studies in naïve rats. This was followed by a comprehensive analysis of immune cells in blood, healthy tissue of the brain, and tumor microenvironment by flow cytometry. RESULTS: Proton FLASH-RT spared memory impairment produced by conventional high-dose proton therapy and induced a similar tumor infiltrating lymphocyte recruitment. Additionally, a general neuroinflammation that was similar in both dose rates was observed. CONCLUSIONS: Overall, this study demonstrated that FLASH proton therapy offers a neuro-protective effect even at high doses while mounting an effective lymphoid immune response in the tumor.

Evaluation of the Role of the Immune System Response After Minibeam Radiation Therapy.

PURPOSE: Minibeam radiation therapy (MBRT) is an innovative technique that uses a spatial dose modulation. The dose distribution consists of high doses (peaks) in the path of the minibeam and low doses (valleys). The underlying biological mechanism associated with MBRT efficacy remains currently unclear and thus we investigated the potential role of the immune system after treatment with MBRT. METHODS AND MATERIALS: Rats bearing an orthotopic glioblastoma cell line were treated with 1 fraction of high dose conventional radiation therapy (30 Gy) or 1 fraction of the same mean dose in MBRT. Both immunocompetent (F344) and immunodeficient (Nude) rats were analyzed in survival studies. Systemic and intratumoral immune cell population changes were studied with flow cytometry and immunohistochemistry (IHC) 2 and 7 days after the irradiation. RESULTS: The absence of response of Nude rats after MBRT suggested that T cells were key in the mode of action of MBRT. An inflammatory phenotype was observed in the blood 1 week after irradiation compared with conventional irradiation. Tumor immune cell analysis by flow cytometry showed a substantial infiltration of lymphocytes, specifically of CD8 T cells and B cells in both conventional and MBRT-treated animals. IHC revealed that MBRT induced a faster recruitment of CD8 and CD4 T cells. Animals that were cured by radiation therapy did not suffer tumor growth after reimplantation of tumoral cells, proving the long-term immunity response generated after a high dose of radiation. CONCLUSIONS: Our findings show that MBRT can elicit a robust antitumor immune response in glioblastoma while avoiding the high toxicity of a high dose of conventional radiation therapy.

Magnetic resonance elastography with guided pressure waves.

Magnetic resonance elastography aims to non-invasively and remotely characterize the mechanical properties of living tissues. To quantitatively and regionally map the shear viscoelastic moduli in vivo, the technique must achieve proper mechanical excitation throughout the targeted tissues. Although it is straightforward, ante manibus, in close organs such as the liver or the breast, which practitioners clinically palpate already, it is somewhat fortunately highly challenging to trick the natural protective barriers of remote organs such as the brain. So far, mechanical waves have been induced in the latter by shaking the surrounding cranial bones. Here, the skull was circumvented by guiding pressure waves inside the subject's buccal cavity so mechanical waves could propagate from within through the brainstem up to the brain. Repeatable, reproducible and robust displacement fields were recorded in phantoms and in vivo by magnetic resonance elastography with guided pressure waves such that quantitative mechanical outcomes were extracted in the human brain.

Glycemic response, satiety, gastric secretions and emptying after bread consumption with water, tea or lemon juice: a randomized crossover intervention using MRI.

PURPOSE: Numerous studies, including our previous work with lemon juice, have reported that low-pH meals reduce the glycemic response to starchy foods. However, the underlying mechanism is not yet understood. Tea, for its polyphenol content, has also been investigated. The main objective of this research was to concurrently study gastric emptying, appetite perceptions and glycemic responses to bread consumed with water, tea, or lemon juice. METHODS: In this randomized, crossover intervention, ten participants consumed equal portions of bread (100 g) with 250 mL of water, water-diluted lemon juice, or black tea at breakfast. Gastric volumes, blood glucose concentrations and appetite perceptions were alternately assessed over 180 min using magnetic resonance imaging, the finger-prick method and visual analogue scales, respectively. RESULTS: Compared to water, lemon juice led to a 1.5 fold increase of the volume of gastric contents, 30 min after the meal (454.0 ± 18.6 vs. 298.4 ± 19.5 mL, [Formula: see text] ± SEM P < 0.00001). Gastric emptying was also 1.5 times faster (P < 0.01). Conversely, lemon juice elicited a lower glycemic response than water (blood glucose concentrations at t = 55 min were 35% lower, P = 0.039). Tea had no effect. Changes in appetite perceptions and gastric volumes correlated well, but with no significant differences between the meals. CONCLUSIONS: Lemon juice lowered the glycemic response and increased both gastric secretions and emptying rate. The results are compatible with the hypothesis that the reduction of the glycemic response is mainly due to the interruption of starch hydrolysis via the acid-inhibition of salivary α-amylase. TRIAL REGISTRATION NUMBER: NCT03265392, August 29, 2017.

First Evaluation of Temporal and Spatial Fractionation in Proton Minibeam Radiation Therapy of Glioma-Bearing Rats.

(1) Background: Proton minibeam radiation therapy (pMBRT) is a new radiotherapy technique using spatially modulated narrow proton beams. pMBRT results in a significantly reduced local tissue toxicity while maintaining or even increasing the tumor control efficacy as compared to conventional radiotherapy in small animal experiments. In all the experiments performed up to date in tumor bearing animals, the dose was delivered in one single fraction. This is the first assessment on the impact of a temporal fractionation scheme on the response of glioma-bearing animals to pMBRT. (2) Methods: glioma-bearing rats were irradiated with pMBRT using a crossfire geometry. The response of the irradiated animals in one and two fractions was compared. An additional group of animals was also treated with conventional broad beam irradiations. (3) Results: pMBRT delivered in two fractions at the biological equivalent dose corresponding to one fraction resulted in the highest median survival time, with 80% long-term survivors free of tumors. No increase in local toxicity was noted in this group with respect to the other pMBRT irradiated groups. Conventional broad beam irradiations resulted in the most severe local toxicity. (4) Conclusion: Temporal fractionation increases the therapeutic index in pMBRT and could ease the path towards clinical trials.

X-rays minibeam radiation therapy at a conventional irradiator: Pilot evaluation in F98-glioma bearing rats and dose calculations in a human phantom.

Minibeam radiation therapy (MBRT) is a type of spatial fractionated radiotherapy that uses submillimetric beams. This work reports on a pilot study on normal tissue response and the increase of the lifespan of glioma-bearing rats when irradiated with a tabletop x-ray system. Our results show a significant widening of the therapeutic window for brain tumours treated with MBRT: an important proportion of long-term survivals (60%) coupled with a significant reduction of toxicity when compared with conventional (broad beam) irradiations. In addition, the clinical translation of the minibeam treatment at a conventional irradiator is evaluated through a possible human head treatment plan.

Evaluation of capacitive micromachined ultrasonic transducers for passive monitoring of microbubble-assisted ultrasound therapies.

Passive cavitation detection can be performed to monitor microbubble activity during brain therapy. Microbubbles under ultrasound exposure generate a response characterized by multiple nonlinear emissions. Here, the wide bandwidth of capacitive micromachined ultrasonic transducers (CMUTs) was exploited to monitor the microbubble signature through a rat skull and a macaque skull. The intrinsic nonlinearity of the CMUTs was characterized in receive mode. Indeed, undesirable nonlinear components generated by the CMUTs must be minimized as they can mask the microbubble harmonic response. The microbubble signature at harmonic and ultra-harmonic components (0.5-6 MHz) was successfully extracted through a rat skull using moderate bias voltage.

Spatially Modulated Proton Minibeams Results in the Same Increase of Lifespan as a Uniform Target Dose Coverage in F98-Glioma-Bearing Rats.

Proton minibeam radiation therapy (pMBRT) is a new approach in proton radiotherapy, by which a significant increase in the therapeutic index has already been demonstrated in RG2 glioma-bearing rats. In the current study we investigated the response of other types of glioma (F98) and performed a comparative evaluation of tumor control effectiveness by pMBRT (with different levels of dose heterogeneity) versus conventional proton therapy. The results of our study showed an equivalent increase in the lifespan for all evaluated groups (conventional proton irradiation and pMBRT) and no significant differences in the histopathological analysis of the tumors or remaining brain tissue. The reduced long-term toxicity observed with pMBRT in previous evaluations at the same dose suggests a possible use of pMBRT to treat glioma with less side effects while ensuring the same tumor control achieved with standard proton therapy.

A temperature-controlled cryogen free cryostat integrated with transceiver-mode superconducting coil for high-resolution magnetic resonance imaging.

Small-sized High Temperature Superconducting (HTS) radiofrequency coils are used in a number of micro-magnetic resonance imaging applications and demonstrate a high detection sensitivity that improves the signal-to-noise ratio. However, the use of HTS coils could be limited by the rarity of cryostats that are suitable for the MR environment. This study presents a magnetic resonance (MR)-compatible and easily operated cryogen-free cryostat based on the pulse tube cryocooler technology for the cooling and monitoring of HTS coils below the temperature of liquid nitrogen. This cryostat features a real-time temperature control function that allows the precise frequency adjustment of the HTS coil. The influence of the temperature on the electrical properties, resonance frequency (f0), and quality factor (Q) of the HTS coil was investigated. Temperature control is obtained with an accuracy of over 0.55 K from 60 K to 86 K, and the sensitivity of the system, extracted from the frequency measurement from 60 K to 75 K, is of about 2 kHz/K, allowing a fine retuning (within few Hz, compared to 10 kHz bandwidth) in good agreement with experimental requirements. We demonstrated that the cryostat, which is mainly composed of non-magnetic materials, does not perturb the electromagnetic field in any way. MR images of a 10 × 10 × 15 mm3 liquid phantom were acquired using the HTS coil as a transceiver with a spatial resolution of 100 × 100 × 300 µm3 in less than 20 min under experimental conditions at 1.5 T.

Ultrasound Molecular Imaging of Renal Cell Carcinoma: VEGFR targeted therapy monitored with VEGFR1 and FSHR targeted microbubbles.

Recent treatment developments for metastatic renal cell carcinoma offer combinations of immunotherapies or immunotherapy associated with tyrosine kinase inhibitors (TKI). There is currently no argument to choose one solution or another. Easy-to-use markers to assess longitudinal responses to TKI are necessary to determine when to switch to immunotherapies. These new markers will enable an earlier adaptation of therapeutic strategy in order to prevent tumor development, unnecessary toxicity and financial costs. This study evaluates the potential of ultrasound molecular imaging to track the response to sunitinib in a clear cell renal carcinoma model (ccRCC). We used a patient-derived xenograft model for this imaging study. Mice harboring human ccRCC were randomized for sunitinib treatment vs. control. The tumors were imaged at days 0, 7, 14 and 28 with ultrasound molecular imaging. Signal enhancement was quantified and compared between the two groups after injections of non-targeted microbubbles and microbubbles targeting VEGFR1 and FSHR. The tumor growth of the sunitinib group was significantly slower. There was a significantly lower expression of both VEGFR-1 and FSHR molecular ultrasound imaging signals in the sunitinib group at all times of treatment (Days 7, 14 and 28). These results confirm the study hypothesis. There was no significant difference between the 2 groups for the non-targeted microbubble ultrasound signal. This study demonstrated for the first time the potential of VEGFR1 and FSHR, by ultrasound-based molecular imaging, to follow-up the longitudinal response to sunitinib in ccRCC. These results should trigger developments for clinical applications.

Design of a fast field-cycling magnetic resonance imaging system, characterization and methods for relaxation dispersion measurements around 1.5 T.

The dependence of the nuclear magnetic resonance relaxation rate on the magnetic field has been widely studied, in particular, in biomedical areas with the objectives to better understand the underlying microscopic mechanisms in tissues and provide biomarkers of diseases. By combining fast-field cycling (FFC) and magnetic resonance imaging (MRI), it is possible to provide localized relaxation dispersion measurements in heterogeneous systems with recent demonstrations in solutions, biological samples, human beings, and small animals. We report here the developments and performances of a device designed for small animal FFC-MRI comprising a resistive insert technology operating inside a 1.5 T MRI system. Specific measurement methods were developed to characterize the system efficiency, response time, homogeneity, stability, and compensation. By adding a non-linear element in the system and using a dual amplifier strategy, it is shown that large field offsets can be produced during relaxation periods while maintaining precise field control during detection periods. The measurement of longitudinal nuclear magnetic relaxation dispersion (NMRD) profiles in the range of 1.08 T-1.92 T is reported, essentially displaying a linear variation in this range for common MRI contrast agents. The slopes of both the longitudinal and transverse relaxation dispersion profiles at 1.5 T are measured and validated, extending the capabilities of previous approaches. The performances of a longitudinal relaxation dispersion mapping method are finally reported, opening the way to quantitative preclinical dispersion imaging studies at a high FFC-MRI field.

Multimodal imaging for tumour characterization from micro- to macroscopic level using a newly developed dorsal chamber designed for long-term follow-up.

Optical imaging of living animals is a unique method of studying the dynamics of physiological and pathological processes at a subcellular level. One-shot acquisitions at high resolution can be achieved on exteriorized organs before animal euthanasia. For longitudinal follow-up, intravital imaging can be used and involves imaging windows implanted in cranial, thoracic or dorsal regions. Several imaging window models exist, but none have proven to be applicable for long-term monitoring and most biological processes take place over several weeks. Moreover, none are compatible with multiple imaging modalities, meaning that different biological parameters cannot be assessed in an individual animal. We developed a new dorsal chamber that was well tolerated by mice (over several months) and allowed individual and collective cell tracking and behaviour analysis by optical imaging, ultrasound and magnetic resonance tomography. This new model broadens potential applications to areas requiring study of long-term biological processes, as in cancer research.

Proton minibeam radiation therapy widens the therapeutic index for high-grade gliomas.

Proton minibeam radiation therapy (pMBRT) is a novel strategy which has already shown a remarkable reduction in neurotoxicity as to compared with standard proton therapy. Here we report on the first evaluation of tumor control effectiveness in glioma bearing rats with highly spatially modulated proton beams. Whole brains (excluding the olfactory bulb) of Fischer 344 rats were irradiated. Four groups of animals were considered: a control group (RG2 tumor bearing rats), a second group of RG2 tumor-bearing rats and a third group of normal rats that received pMBRT (70 Gy peak dose in one fraction) with very heterogeneous dose distributions, and a control group of normal rats. The tumor-bearing and normal animals were followed-up for 6 months and one year, respectively. pMBRT leads to a significant tumor control and tumor eradication in 22% of the cases. No substantial brain damage which confirms the widening of the therapeutic window for high-grade gliomas offered by pMBRT. Additionally, the fact that large areas of the brain can be irradiated with pMBRT without significant side effects, would allow facing the infiltrative nature of gliomas.

Proton minibeam radiation therapy spares normal rat brain: Long-Term Clinical, Radiological and Histopathological Analysis.

Proton minibeam radiation therapy (pMBRT) is a novel strategy for minimizing normal tissue damage resulting from radiotherapy treatments. This strategy partners the inherent advantages of protons for radiotherapy with the gain in normal tissue preservation observed upon irradiation with narrow, spatially fractionated beams. In this study, whole brains (excluding the olfactory bulb) of Fischer 344 rats (n = 16) were irradiated at the Orsay Proton Therapy Center. Half of the animals received standard proton irradiation, while the other half were irradiated with pMBRT at the same average dose (25 Gy in one fraction). The animals were followed-up for 6 months. A magnetic resonance imaging (MRI) study using a 7-T small-animal MRI scanner was performed along with a histological analysis. Rats treated with conventional proton irradiation exhibited severe moist desquamation, permanent epilation and substantial brain damage. In contrast, rats in the pMBRT group exhibited no skin damage, reversible epilation and significantly reduced brain damage; some brain damage was observed in only one out of the eight irradiated rats. These results demonstrate that pMBRT leads to an increase in normal tissue resistance. This net gain in normal tissue sparing can lead to the efficient treatment of very radio-resistant tumours, which are currently mostly treated palliatively.

Toward a Standardization of Ultrasound Scanners for Dynamic Contrast-Enhanced Ultrasonography: Methodology and Phantoms.

The standardization of ultrasound scanners for dynamic contrast-enhanced ultrasonography (DCE-US) is mandatory for evaluation of clinical multicenter studies. We propose a robust method using a phantom for measuring the variation of the harmonic signal intensity obtained from the area under the time-intensity curve versus various contrast-agent concentrations. The slope of this measured curve is the calibration parameter. We tested our method on two devices from the same manufacturer (AplioXV and Aplio500, Toshiba, Tokyo, Japan) using the same settings as defined for a French multicenter study. The Aplio500's settings were adjusted to match the slopes of the AplioXV, resulting in the following settings on the Aplio500: at 3.5 MHz: MI = 0.15; CG = 35 dB and at 8 MHz: MI = 0.10; CG = 32 dB. This calibration method is very important for future DCE-US multicenter studies.