Democratizing FLASH Radiotherapy.

FLASH radiotherapy (RT) is emerging as a potentially revolutionary advancement in cancer treatment, offering the potential to deliver RT at ultra-high dose rates (>40 Gy/s) while significantly reducing damage to healthy tissues. Democratizing FLASH RT by making this cutting-edge approach more accessible and affordable for healthcare systems worldwide would have a substantial impact in global health. Here, we review recent developments in FLASH RT and present perspective on further developments that could facilitate the democratizing of FLASH RT. These include upgrading and validating current technologies that can deliver and measure the FLASH radiation dose with high accuracy and precision, establishing a deeper mechanistic understanding of the FLASH effect, and optimizing dose delivery conditions and parameters for different types of tumors and normal tissues, such as the dose rate, dose fractionation, and beam quality for high efficacy. Furthermore, we examine the potential for democratizing FLASH radioimmunotherapy leveraging evidence that FLASH RT can make the tumor microenvironment more immunogenic, and parallel developments in nanomedicine or use of smart radiotherapy biomaterials for combining RT and immunotherapy. We conclude that the democratization of FLASH radiotherapy represents a major opportunity for concerted cross-disciplinary research collaborations with potential for tremendous impact in reducing radiotherapy disparities and extending the cancer moonshot globally.

Feasibility of Synchrotron-Based Ultra-High Dose Rate (UHDR) Proton Irradiation with Pencil Beam Scanning for FLASH Research.

BACKGROUND: This study aims to present the feasibility of developing a synchrotron-based proton ultra-high dose rate (UHDR) pencil beam scanning (PBS) system. METHODS: The RF extraction power in the synchrotron system was increased to generate 142.4 MeV pulsed proton beams for UHDR irradiation at ~100 nA beam current. The charge per spill was measured using a Faraday cup. The spill length and microscopic time structure of each spill was measured with a 2D strip transmission ion chamber. The measured UHDR beam fluence was used to derive the spot dwell time for pencil beam scanning. Absolute dose distributions at various depths and spot spacings were measured using Gafchromic films in a solid-water phantom. RESULTS: For proton UHDR beams at 142.4 MeV, the maximum charge per spill is 4.96 ± 0.10 nC with a maximum spill length of 50 ms. This translates to an average beam current of approximately 100 nA during each spill. Using a 2 × 2 spot delivery pattern, the delivered dose per spill at 5 cm and 13.5 cm depth is 36.3 Gy (726.3 Gy/s) and 56.2 Gy (1124.0 Gy/s), respectively. CONCLUSIONS: The synchrotron-based proton therapy system has the capability to deliver pulsed proton UHDR PBS beams. The maximum deliverable dose and field size per pulse are limited by the spill length and extraction charge.

FLEX: FLexible Transducer With External Tracking for Ultrasound Imaging With Patient-Specific Geometry Estimation.

Flexible array transducers can adapt to patient-specific geometries during real-time ultrasound (US) image-guided therapy monitoring. This makes the system radiation-free and less user-dependency. Precise estimation of the flexible transducer's geometry is crucial for the delay-and-sum (DAS) beamforming algorithm to reconstruct B-mode US images. The primary innovation of this research is to build a system named FLexible transducer with EXternal tracking (FLEX) to estimate the position of each element of the flexible transducer and reconstruct precise US images. FLEX utilizes customized optical markers and a tracker to monitor the probe's geometry, employing a polygon fitting algorithm to estimate the position and azimuth angle of each transducer element. Subsequently, the traditional DAS algorithm processes the delay estimation from the tracked element position, reconstructing US images from radio-frequency (RF) channel data. The proposed method underwent evaluation on phantoms and cadaveric specimens, demonstrating its clinical feasibility. Deviations in tracked probe geometry compared to ground truth were minimal, measuring 0.50 ± 0.29 mm for the CIRS phantom, 0.54 ± 0.35 mm for the deformable phantom, and 0.36 ± 0.24 mm on the cadaveric specimen. Reconstructing the US image using tracked probe geometry significantly outperformed the untracked geometry, as indicated by a Dice score of 95.1 ± 3.3% versus 62.3 ± 9.2% for the CIRS phantom. The proposed method achieved high accuracy (<0.5 mm error) in tracking the element position for various random curvatures applicable for clinical deployment. The evaluation results show that the radiation-free proposed method can effectively reconstruct US images and assist in monitoring image-guided therapy with minimal user dependency.

Dosimetric characterization of a rotating anode x-ray tube for FLASH radiotherapy research.

PURPOSE: Most current research toward ultra-high dose rate (FLASH) radiation is conducted with advanced proton and electron accelerators, which are of limited accessibility to basic laboratory research. An economical alternative to charged particle accelerators is to employ high-capacity rotating anode x-ray tubes to produce kilovoltage x-rays at FLASH dose rates at short source-to-surface distances (SSD). This work describes a comprehensive dosimetric evaluation of a rotating anode x-ray tube for potential application in laboratory FLASH study. METHODS AND MATERIALS: A commercially available high-capacity fluoroscopy x-ray tube with 75 kW input power was implemented as a potential FLASH irradiator. Radiochromic EBT3 film and thermoluminescent dosimeters (TLDs) were used to investigate the effects of SSD and field size on dose rates and depth-dose characteristics in kV-compatible solid water phantoms. Custom 3D printed accessories were developed to enable reproducible phantom setup at very short SSD. Open and collimated radiation fields were assessed. RESULTS: Despite the lower x-ray energy and short SSD used, FLASH dose rates above 40 Gy/s were achieved for targets up to 10-mm depth in solid water. Maximum surface dose rates of 96 Gy/s were measured in the open field at 47 mm SSD. A non-uniform high-to-low dose gradient was observed in the planar dose distribution, characteristic of anode heel effects. With added collimation, beams up to 10-mm diameter with reasonable uniformity can be produced. Typical 80%-20% penumbra in the collimated x-ray FLASH beams were less than 1 mm at 5-mm depth in phantom. Ramp-up times at the maximum input current were less than 1 ms. CONCLUSION: Our dosimetric characterization demonstrates that rotating anode x-ray tube technology is capable of producing radiation beams in support of preclinical FLASH radiobiology research.

Characterization of a commercial bioluminescence tomography-guided system for pre-clinical radiation research.

BACKGROUND: Widely used Cone-beam computed tomography (CBCT)-guided irradiators have limitations in localizing soft tissue targets growing in a low-contrast environment. This hinders small animal irradiators achieving precise focal irradiation. PURPOSE: To advance image-guidance for soft tissue targeting, we developed a commercial-grade bioluminescence tomography-guided system (BLT, MuriGlo) for pre-clinical radiation research. We characterized the system performance and demonstrated its capability in target localization. We expect this study can provide a comprehensive guideline for the community in utilizing the BLT system for radiation studies. METHODS: MuriGlo consists of four mirrors, filters, lens, and charge-coupled device (CCD) camera, enabling a compact imaging platform and multi-projection and multi-spectral BLT. A newly developed mouse bed allows animals imaged in MuriGlo and transferred to a small animal radiation research platform (SARRP) for CBCT imaging and BLT-guided irradiation. Methods and tools were developed to evaluate the CCD response linearity, minimal detectable signal, focusing, spatial resolution, distortion, and uniformity. A transparent polycarbonate plate covering the middle of the mouse bed was used to support and image animals from underneath the bed. We investigated its effect on 2D Bioluminescence images and 3D BLT reconstruction accuracy, and studied its dosimetric impact along with the rest of mouse bed. A method based on pinhole camera model was developed to map multi-projection bioluminescence images to the object surface generated from CBCT image. The mapped bioluminescence images were used as the input data for the optical reconstruction. To account for free space light propagation from object surface to optical detector, a spectral derivative (SD) method was implemented for BLT reconstruction. We assessed the use of the SD data (ratio imaging of adjacent wavelength) in mitigating out of focusing and non-uniformity seen in the images. A mouse phantom was used to validate the data mapping. The phantom and an in vivo glioblastoma model were utilized to demonstrate the accuracy of the BLT target localization. RESULTS: The CCD response shows good linearity with < 0.6% residual from a linear fit. The minimal detectable level is 972 counts for 10 × 10 binning. The focal plane position is within the range of 13-18 mm above the mouse bed. The spatial resolution of 2D optical imaging is < 0.3 mm at Rayleigh criterion. Within the region of interest, the image uniformity is within 5% variation, and image shift due to distortion is within 0.3 mm. The transparent plate caused < 6% light attenuation. The use of the SD imaging data can effectively mitigate out of focusing, image non-uniformity, and the plate attenuation, to support accurate multi-spectral BLT reconstruction. There is < 0.5% attenuation on dose delivery caused by the bed. The accuracy of data mapping from the 2D bioluminescence images to CBCT image is within 0.7 mm. Our phantom test shows the BLT system can localize a bioluminescent target within 1 mm with an optimal threshold and only 0.2 mm deviation was observed for the case with and without a transparent plate. The same localization accuracy can be maintained for the in vivo GBM model. CONCLUSIONS: This work is the first systematic study in characterizing the commercial BLT-guided system. The information and methods developed will be useful for the community to utilize the imaging system for image-guided radiation research.

FLASH Effects Induced by Orthovoltage X-Rays.

PURPOSE: This work describes the first implementation and in vivo study of ultrahigh-dose-rate radiation (>37 Gy/s; FLASH) effects induced by kilovoltage (kV) x-ray from a rotating-anode x-ray source. METHODS AND MATERIALS: A high-capacity rotating-anode x-ray tube with an 80-kW generator was implemented for preclinical FLASH radiation research. A custom 3-dimensionally printed immobilization and positioning tool was developed for reproducible irradiation of a mouse hind limb. Calibrated Gafchromic (EBT3) film and thermoluminescent dosimeters (LiF:Mg,Ti) were used for in-phantom and in vivo dosimetry. Healthy FVB/N and FVBN/C57BL/6 outbred mice were irradiated on 1 hind leg to doses up to 43 Gy at FLASH (87 Gy/s) and conventional (CONV; <0.05 Gy/s) dose rates. The radiation doses were delivered using a single pulse with the widths up to 500 ms and 15 minutes at FLASH and CONV dose rates. Histologic assessment of radiation-induced skin damage was performed at 8 weeks posttreatment. Tumor growth suppression was assessed using a B16F10 flank tumor model in C57BL6J mice irradiated to 35 Gy at both FLASH and CONV dose rates. RESULTS: FLASH-irradiated mice experienced milder radiation-induced skin injuries than CONV-irradiated mice, visible by 4 weeks posttreatment. At 8 weeks posttreatment, normal tissue injury was significantly reduced in FLASH-irradiated animals compared with CONV-irradiated animals for histologic endpoints including inflammation, ulceration, hyperplasia, and fibrosis. No difference in tumor growth response was observed between FLASH and CONV irradiations at 35 Gy. The normal tissue sparing effects of FLASH irradiations were observed only for high-severity endpoint of ulceration at 43 Gy, which suggests the dependency of biologic endpoints to FLASH radiation dose. CONCLUSIONS: Rotating-anode x-ray sources can achieve FLASH dose rates in a single pulse with dosimetric properties suitable for small-animal experiments. We observed FLASH normal tissue sparing of radiation toxicities in mouse skin irradiated at 35 Gy with no sacrifice to tumor growth suppression. This study highlights an accessible new modality for laboratory study of the FLASH effect.

Quantitative Bioluminescence Tomography for In Vivo Volumetric-Guided Radiotherapy.

Several groups, including ours, have initiated efforts to develop small-animal irradiators that mimic radiation therapy (RT) for human treatment. The major image modality used to guide irradiation is cone-beam computed tomography (CBCT). While CBCT provides excellent guidance capability, it is less adept at localizing soft tissue targets growing in a low image contrast environment. In contrast, bioluminescence imaging (BLI) provides strong image contrast and thus is an attractive solution for soft tissue targeting. However, commonly used 2D BLI on an animal surface is inadequate to guide irradiation, because optical transport from an internal bioluminescent tumor is highly susceptible to the effects of optical path length and tissue absorption and scattering. Recognition of these limitations led us to integrate 3D bioluminescence tomography (BLT) with the small animal radiation research platform (SARRP). In this chapter, we introduce quantitative BLT (QBLT) with the advanced capabilities of quantifying tumor volume for irradiation guidance. The detail of system components, calibration protocol, and step-by-step procedure to conduct the QBLT-guided irradiation are described.

Development of a Mobile Fluorescence Tomography-guided System for Pre-clinical Radiotherapy Research.

We proposed to build a mobile fluorescence tomography (mFT) system as an image-guided platform for pre-clinical radiotherapy research. The mFT system is expected to localize functional target/tumor, guide irradiation, and provide longitudinal treatment assessment.

Hemodynamics in growing and stable cerebral aneurysms.

OBJECTIVE: The detailed mechanisms of cerebral aneurysm evolution are poorly understood but are important for objective aneurysm evaluation and improved patient management. The purpose of this study was to identify hemodynamic conditions that may predispose aneurysms to growth. METHODS: A total of 33 intracranial unruptured aneurysms longitudinally followed with three-dimensional imaging were studied. Patient-specific computational fluid dynamics models were constructed and used to quantitatively characterize the hemodynamic environments of these aneurysms. Hemodynamic characteristics of growing (n=16) and stable (n=17) aneurysms were compared. Logistic regression statistical models were constructed to test the predictability of aneurysm growth by hemodynamic features. RESULTS: Growing aneurysms had significantly smaller shear rate ratios (p=0.01), higher concentration of wall shear stress (p=0.03), smaller vorticity ratios (p=0.01), and smaller viscous dissipation ratios (p=0.01) than stable aneurysms. They also tended to have larger areas under low wall shear stress (p=0.06) and larger aspect ratios (p=0.18), but these trends were not significant. Mean wall shear stress was not significantly different between growing and stable aneurysms. Logistic regression models based on hemodynamic variables were able to discriminate between growing and stable aneurysms with a high degree of accuracy (94-100%). CONCLUSIONS: Growing aneurysms tend to have complex intrasaccular flow patterns that induce non-uniform wall shear stress distributions with areas of concentrated high wall shear stress and large areas of low wall shear stress. Statistical models based on hemodynamic features seem capable of discriminating between growing and stable aneurysms.

Analysis of hemodynamics and wall mechanics at sites of cerebral aneurysm rupture.

BACKGROUND: It is thought that aneurysms evolve as the result of progressive degradation of the wall in response to abnormal hemodynamics characterized by either high or low wall shear stress (WSS). OBJECTIVE: To investigate the effects of these two different hemodynamic pathways in a series of cerebral aneurysms with known rupture sites. METHODS: Nine aneurysms in which the rupture site could be identified in three-dimensional images were analyzed. The WSS distribution was obtained from computational fluid dynamics (CFD) simulations. Internal wall stresses were computed using structural wall models under hemodynamic loads determined by the CFD models. Wall properties (thickness and stiffness) were modulated with the WSS distribution (increased or decreased in regions of high or low WSS) to test possible wall degradation pathways. Rupture probability indices (RPI) were calculated to compare different wall models. RESULTS: Most rupture sites aligned with the intrasaccular flow stream and downstream of the primary impaction zone. The model that best explained the rupture site (produced higher RPI) in eight of the nine aneurysms (89%) had thinner and stiffer walls in regions of abnormally high WSS. The remaining case (11%) was best explained by a model with thinner and stiffer walls in regions of abnormally low WSS. CONCLUSIONS: Aneurysm rupture seems to be caused by localized degradation and weakening of the wall in response to abnormal hemodynamics. Image-based computational models assuming wall thinning and stiffening in regions of abnormally high WSS were able to explain most of the observed rupture sites.

Computational fluid dynamics in brain aneurysms.

Because of its ability to deal with any geometry, image-based computational fluid dynamics (CFD) has been progressively used to investigate the role of hemodynamics in the underlying mechanisms governing the natural history of cerebral aneurysms. Despite great progress in methodological developments and many studies using patient-specific data, there are still significant controversies about the precise governing processes and divergent conclusions from apparently contradictory results. Sorting out these issues requires a global vision of the state of the art and a unified approach to solving this important scientific problem. Towards this end, this paper reviews the contributions made using patient-specific CFD models to further the understanding of these mechanisms, and highlights the great potential of patient-specific computational models for clinical use in the assessment of aneurysm rupture risk and patient management.

Hemodynamic Analysis of Intracranial Aneurysms with Moving Parent Arteries: Basilar Tip Aneurysms.

The effects of parent artery motion on the hemodynamics of basilar tip saccular aneurysms and its potential effect on aneurysm rupture were studied.The aneurysm and parent artery motions in two patients were determined from cine loops of dynamic angiographies. The oscillatory motion amplitude was quantified by registering the frames. Patient-specific computational fluid dynamics (CFD) models of both aneurysms were constructed from 3D rotational angiography images. Two CFD calculations were performed for each patient, corresponding to static and moving models. The motion estimated from the dynamic images was used to move the surface grid points in the moving model. Visualizations from the simulations were compared for wall shear stress (WSS), velocity profiles, and streamlines.In both patients a rigid oscillation of the aneurysm and basilar artery in the anterio-posterior direction was observed and measured. The distribution of WSS was nearly identical between the models of each patient, as well as major intra-aneurysmal flow structures, inflow jets, and regions of impingement.The motion observed in pulsating intracranial vasculature does not have a major impact on intra-aneurysmal hemodynamic variables. Parent artery motion is unlikely to be a risk factor for increased risk of aneurysmal rupture.

Hemodynamics of Cerebral Aneurysms.

The initiation and progression of cerebral aneurysms are degenerative processes of the arterial wall driven by a complex interaction of biological and hemodynamic factors. Endothelial cells on the artery wall respond physiologically to blood-flow patterns. In normal conditions, these responses are associated with nonpathological tissue remodeling and adaptation. The combination of abnormal blood patterns and genetics predisposition could lead to the pathological formation of aneurysms. Here, we review recent progress on the basic mechanisms of aneurysm formation and evolution, with a focus on the role of hemodynamic patterns.

Changes in global gene expression in rat myometrium in transition from late pregnancy to parturition.

The process of parturition involves the complex interplay of factors that change the excitability and contractile activity of the uterus. We have compared the relative gene expression profile of myometrium from rats before parturition (21 days pregnant) and during delivery, using high-density DNA microarray. Of 8,740 sequences available in the array, a total of 3,782 were detected as present. From the sequences that were significantly altered, 59 genes were upregulated and 82 genes were downregulated. We were able to detect changes in genes described to have altered expression level at term, including connexin 43 and 26, cyclooxygenase 2, and oxytocin receptor, as well as novel genes that have been not previously associated with parturition. Quantitative real-time PCR on selected genes further confirmed the microarray data. Here we report for the first time that aquaporin5 (AQP5), a member of the aquaporin water channel family, was dramatically downregulated during parturition (approximately 100-fold by microarray and approximately 50-fold by real-time PCR). The emerging profile highlights biochemical cascades occurring in a period of approximately 36 h that trigger parturition and the initiation of myometrium reverse remodeling postpartum. The microarray analysis uncovered genes that were previously suspected to play a role in parturition. This regulation involves genes from immune/inflammatory response, steroid/lipid metabolism, calcium homeostasis, cell volume regulation, cell signaling, cell division, and tissue remodeling, suggesting the presence of multiple and redundant mechanisms altered in the process of birth.

Mitochondrial dysfunction, oxidative stress, and apoptosis revealed by proteomic and transcriptomic analyses of the striata in two mouse models of Parkinson's disease.

The molecular mechanisms underlying the changes in the nigrostriatal pathway in Parkinson's disease (PD) are not completely understood. Here, we use mass spectrometry and microarrays to study the proteomic and transcriptomic changes in the striatum of two mouse models of PD, induced by the distinct neurotoxins 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and methamphetamine (METH). Proteomic analyses resulted in the identification and relative quantification of 912 proteins with two or more unique peptides and 86 proteins with significant abundance changes following neurotoxin treatment. Similarly, microarray analyses revealed 181 genes with significant changes in mRNA, following neurotoxin treatment. The combined protein and gene list provides a clearer picture of the potential mechanisms underlying neurodegeneration observed in PD. Functional analysis of this combined list revealed a number of significant categories, including mitochondrial dysfunction, oxidative stress response, and apoptosis. These results constitute one of the largest descriptive data sets integrating protein and transcript changes for these neurotoxin models with many similar end point phenotypes but distinct mechanisms.

Genetic program of neuronal differentiation and growth induced by specific activation of NMDA receptors.

Glutamate and its receptors are expressed very early during development and may play important roles in neurogenesis, synapse formation and brain wiring. The levels of glutamate and activity of its receptors can be influenced by exogenous factors, leading to neurodevelopmental disorders. To investigate the role of NMDA receptors on gene regulation in a neuronal model, we used primary neuronal cultures developed from embryonic rat cerebri in serum-free medium. Using Affymetrix Gene Arrays, we found that genes known to be involved in neuronal plasticity were differentially expressed 24 h after a brief activation of NMDA receptors. The upregulation of these genes was accompanied by a sustained induction of CREB phosphorylation, and an increase in synaptophysin immunoreactivity. We conclude that NMDA receptor activation elicits expression of genes whose downstream products are involved in the regulation of early phases of the process leading to synaptogenesis and its consolidation, at least in part through sustained CREB phosphorylation.

Gene expression is differentially regulated by neurotransmitters in embryonic neuronal cortical culture.

Neurotransmitters and their receptors have been involved in both proper brain development and neurodevelopmental disorders. The role that nicotinic receptors play in immature cortical neurons was initially investigated by gene profiling using Affymetrix DNA arrays. Both short (15 min) and prolonged (18 h) treatments with nicotine did not induce modification in gene expression, whereas a significant down-regulation of c-fos protein levels was observed after 18 h treatment. Conversely, a brief treatment with the glutamatergic agonist NMDA triggered up-regulation of immediate early genes and transcription factors, which remained unaffected by pre-treatment for 18 h with nicotine. Calcium imaging studies revealed that NMDA activated a sustained increase in intracellular calcium concentration in the majority of neurons, whereas nicotine evoked only a transient calcium increase in a smaller percentage of neurons, suggesting that the calcium signalling response was correlated with activation of gene expression. Nicotine effects on immature cortical neurons perhaps do not require gene regulation but may be still acting on signalling pathways.

Development and evaluation of a micro- and nanoscale proteomic sample preparation method.

Challenges associated with the efficient and effective preparation of micro- and nanoscale (micro- and nanogram) clinical specimens for proteomic applications include the unmitigated sample losses that occur during the processing steps. Herein, we describe a simple "single-tube" preparation protocol appropriate for small proteomic samples using the organic cosolvent, trifluoroethanol (TFE) that circumvents the loss of sample by facilitating both protein extraction and protein denaturation without requiring a separate cleanup step. The performance of the TFE-based method was initially evaluated by comparisons to traditional detergent-based methods on relatively large scale sample processing using human breast cancer cells and mouse brain tissue. The results demonstrated that the TFE-based protocol provided comparable results to the traditional detergent-based protocols for larger, conventionally sized proteomic samples (>100 microg protein content), based on both sample recovery and numbers of peptide/protein identifications. The effectiveness of this protocol for micro- and nanoscale sample processing was then evaluated for the extraction of proteins/peptides and shown effective for small mouse brain tissue samples (approximately 30 microg total protein content) and also for samples of approximately 5000 MCF-7 human breast cancer cells (approximately 500 ng total protein content), where the detergent-based methods were ineffective due to losses during cleanup and transfer steps.

Anatomical methods for voxelation of the mammalian brain.

Voxelation allows high-throughput acquisition of three-dimensional gene expression patterns in the brain through analysis of spatially registered voxels (cubes). The method results in multiple volumetric maps of gene expression analogous to the images reconstructed in biomedical imaging techniques. An important issue for voxelation is the development of approaches to anchor correctly harvested voxels to the underlying anatomy. Here, we describe experiments to identify fixation and cryopreservation protocols for improved registration of harvested voxels with neuroanatomical structures. Paraformaldehyde fixation greatly reduced RNA recovery as judged by ribosomal RNA abundance. However, gene expression signals from paraformaldehyde-fixed samples were not appreciably diminished as judged by average signal-noise ratios from microarrays, highlighting the difficulties of accurate quantitation of cross-linked RNA. Additional use of cryoprotection helped to improve further RNA recovery and signal from fixed tissue. It appears that the best protocol to provide the necessary resolution of neuroanatomical information in voxelation entails a controlled dose of fixation and thorough cryoprotection, complemented by histological staining.