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Monte Carlo (MC) based treatment planning solutions for FLASH have typically been confined within proprietary treatment planning systems (TPS), lacking a true open-source implementation. This study aims to establish an open-source treatment planning workflow by integrating the Tool for Particle Simulation (TOPAS) MC code with a Python graphical user interface (GUI) to accurately calculate 3D dose across various electron cut-out apertures and geometries. The treatment head of the FLASH-enabled Linac was modeled using CAD files. Depth dose and beam profiles were acquired using radiographic film with different circular collimators. A beam model was created through inverse Bayesian optimization, minimizing the difference between measured and simulated profiles. The total particle count for each radiation pulse passing through the beam current transformer (BCT) on the mobile electron linear accelerator was estimated from measurements and then the BCT was integrated into the MC simulation, establishing a calibration relationship between MC output, absolute dose and number of histories. The whole workflow was then used for end-to-end testing incorporating an anatomically correct 3D printed mouse phantom. Inverse optimization yielded a beam energy of 8.4 MeV with a spread of 1 MeV and a source spot size of 2 mm with an angular spread of 4 degrees. The simulated and measured depth dose for the 10 cm collimator agreed to within <1% of each other. Simulated beam profiles for various insert sizes exhibited good agreement (<3%) with the measured profiles, although variations were noted in the low dose region for all profiles. A Python GUI was developed enabling the importation of CT scans and beam apertures to calculate 3D dose distributions by invoking the TOPAS MC code for simulation. A recent multi-institutional dosimetric audit uncovered discrepancies of up to 10%, particularly in areas with tissue inhomogeneities. Given the tissue-protective benefits of FLASH radiation are on the order of 10-20%, it is imperative to employ precise planning and delivery tools. Our open-source MC beam model and Python GUI will be invaluable resources for ensuring accuracy in FLASH research, with the potential for seamless adaptation into clinical trials. [ABSTRACT FROM AUTHOR]

Commercial devices capable of delivering FLASH radiotherapy are being introduced into radiobiology research. There is a critical necessity for a unified dosimetric protocol to ensure dosimetric consistency across institutes. This work introduces a collaborative, multi-institutional dosimetry protocol designed to validate and standardize dosimetry across two research facilities using FLASH intraoperative systems. Two independent institutes used the same collimator design (4x4 cm2) and same beam parameters to deliver 12 or 14 Gy with FLASH or conventional (CONV) dose rates. For dosimetry, replicates of a realistic anatomy 3D-printed mouse phantom with coronal or sagittal division allowing insertions of radiochromic films, and a set of films (sourced from a singular source) were sent from Institute 1 to Institute 2. The institutes independently calibrated the target doses for FLASH using films from the phantom against the associated measurements from the embedded toroid of the system, and for CONV against the total number of MU. Ultimately, 80 experimental toroid measurements for FLASH (40 per target dose) were acquired in 4 days, with 5 set of films per modality per day of measurement. The films were returned to Institute 1 for analysis and the experimental values for FLASH and CONV were calculated. For 12 Gy target dose, the toroid-derived FLASH doses combined for Institute 1 vs Institute 2 were 11.85 ± 0.11 Gy vs 12.32 ± 0.16 Gy, for 14 Gy were 14.12 ± 0.07 Gy vs 14.09 ± 0.01 Gy. for CONV 11.74 ± 0.23 Gy vs 12.19 ± 0.20 Gy and for 14 Gy were 13.86 ± 0.21 Gy vs 14.47 ± 0.31 Gy. The differences between measured and target doses, as well as between FLASH and CONV doses, were within 3%, and the inter-institutional dose differences were under 5%. Enhancing the protocol could entail incorporating an extra calibration step before the experimental dates to fine tune dose alignment between institutions. The success of the collaborative, multi-institutional dosimetry protocol, using realistic anatomy 3D-printed mouse phantom, is evidenced by the demonstrated consistency in preclinical radiobiological experiments across distinct research facilities. [ABSTRACT FROM AUTHOR]

Strategies to enhance the anti-tumor immune response of stereotactic ablative radiotherapy (SABR) at primary tumors and abscopal sites are under intensive investigation. Here we report a metabolizable binary supracluster (BSC gal ) that combines gold nanoclusters as radiosensitizing adjuvants with small interfering RNA (siRNA) targeting the immunosuppressive mediator galectin-1 (Gal-1). BSC gal comprises reversibly crosslinked cationic gold nanoclusters and siRNA complexes in a polymer matrix that biodegrades over weeks, facilitating clearance (90.3% in vivo clearance at 4 weeks) to reduce toxicity. The particle size well above the renal filtration threshold facilitates passive delivery to tumors. Using mouse models of head and neck cancer, we show that BSC gal augments the radiodynamic and immunotherapeutic effects of SABR at the primary and metastatic tumors by promoting tumor-inhibitory leukocytes, upregulating cytotoxic granzyme B and reducing immunosuppressive cell populations. It outperforms SABR plus Gal-1 antagonists, chemoradiation drug cisplatin or PD-1 inhibitor. This work presents a translatable strategy to converge focal radiosensitization with targeted immune checkpoint silencing for personalized radioimmunotherapy., (© 2024. The Author(s), under exclusive licence to Springer Nature America, Inc.)

Background: FLASH radiation therapy (RT) offers a promising avenue for the broadening of the therapeutic index. However, to leverage the full potential of FLASH in the clinical setting, an improved understanding of the biological principles involved is critical. This requires the availability of specialized equipment optimized for the delivery of conventional (CONV) and ultra-high dose rate (UHDR) irradiation for preclinical studies. One method to conduct such preclinical radiobiological research involves adapting a clinical linear accelerator configured to deliver both CONV and UHDR irradiation., Purpose: We characterized the dosimetric properties of a clinical linear accelerator configured to deliver ultra-high dose rate irradiation to two anatomic sites in mice and for cell-culture FLASH radiobiology experiments., Methods: Delivered doses of UHDR electron beams were controlled by a microcontroller and relay interfaced with the respiratory gating system. We also produced beam collimators with indexed stereotactic mouse positioning devices to provide anatomically specific preclinical treatments. Treatment delivery was monitored directly with an ionization chamber, and charge measurements were correlated with radiochromic film measurements at the entry surface of the mice. The setup for conventional dose rate irradiation utilized the same collimation system but at increased source-to-surface distance. Monte Carlo simulations and film dosimetry were used to characterize beam properties and dose distributions., Results: The mean electron beam energies before the flattening filter were 18.8 MeV (UHDR) and 17.7 MeV (CONV), with corresponding values at the mouse surface of 17.2 and 16.2 MeV. The charges measured with an external ion chamber were linearly correlated with the mouse entrance dose. The use of relay gating for pulse control initially led to a delivery failure rate of 20% (± 1 pulse); adjustments to account for the linac latency improved this rate to < 1/20. Beam field sizes for two anatomically specific mouse collimators (4 × 4 cm 2 for whole-abdomen and 1.5 × 1.5 cm 2 for unilateral lung irradiation) were accurate within < 5% and had low radiation leakage (< 4%). Normalizing the dose at the center of the mouse (∼0.75 cm depth) produced UHDR and CONV doses to the irradiated volumes with > 95% agreement., Conclusion: We successfully configured a clinical linear accelerator for increased output and developed a robust preclinical platform for anatomically specific irradiation, with highly accurate and precise temporal and spatial dose delivery, for both CONV and UHDR irradiation applications., (© 2024 American Association of Physicists in Medicine.)

Background and Purpose: Ultra-high dose-rate radiotherapy (FLASH) has been shown to mitigate normal tissue toxicities associated with conventional dose rate radiotherapy (CONV) without compromising tumor killing in preclinical models. A prominent challenge in preclinical radiation research, including FLASH, is validating both the physical dosimetry and the biological effects across multiple institutions., Materials and Methods: We previously demonstrated dosimetric reproducibility of two different electron FLASH devices at separate institutions using standardized phantoms and dosimeters. In this study, tumor-free adult female mice were given 10 Gy whole brain FLASH and CONV irradiation at both institutions and evaluated for the reproducibility and temporal evolution of multiple neurobiological endpoints., Results: FLASH sparing of behavioral performance on novel object recognition (4 months post-irradiation) and of electrophysiologic long-term potentiation (LTP, 5 months post-irradiation) was reproduced between institutions. Differences between FLASH and CONV on the endpoints of hippocampal neurogenesis (Sox2, doublecortin), neuroinflammation (microglial activation), and electrophysiology (LTP) were not observed at early times (48 h to 2 weeks), but recovery of immature neurons by 3 weeks was greater with FLASH., Conclusion: In summary, we demonstrated reproducible FLASH sparing effects on the brain between two different beams at two different institutions with validated dosimetry. FLASH sparing effects on the endpoints evaluated manifested at later but not the earliest time points., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier B.V. All rights reserved.)

Purpose: We conducted a multi-institutional dosimetric audit between FLASH and conventional dose rate (CONV) electron irradiations by using an anatomically realistic 3-dimensional (3D) printed mouse phantom., Methods and Materials: A computed tomography (CT) scan of a live mouse was used to create a 3D model of bony anatomy, lungs, and soft tissue. A dual-nozzle 3D printer was used to print the mouse phantom using acrylonitrile butadiene styrene (∼1.02 g/cm 3 ) and polylactic acid (∼1.24 g/cm 3 ) simultaneously to simulate soft tissue and bone densities, respectively. The lungs were printed separately using lightweight polylactic acid (∼0.64 g/cm 3 ). Hounsfield units (HU), densities, and print-to-print stability of the phantoms were assessed. Three institutions were each provided a phantom and each institution performed 2 replicates of irradiations at selected anatomic regions. The average dose difference between FLASH and CONV dose distributions and deviation from the prescribed dose were measured with radiochromic film., Results: Compared with the reference CT scan, CT scans of the phantom demonstrated mass density differences of 0.10 g/cm 3 for bone, 0.12 g/cm 3 for lung, and 0.03 g/cm 3 for soft tissue regions. Differences in HU between phantoms were <10 HU for soft tissue and bone, with lung showing the most variation (54 HU), but with minimal effect on dose distribution (<0.5%). Mean differences between FLASH and CONV decreased from the first to the second replicate (4.3%-1.2%), and differences from the prescribed dose decreased for both CONV (3.6%-2.5%) and FLASH (6.4%-2.7%). Total dose accuracy suggests consistent pulse dose and pulse number, although these were not specifically assessed. Positioning variability was observed, likely due to the absence of robust positioning aids or image guidance., Conclusions: This study marks the first dosimetric audit for FLASH using a nonhomogeneous phantom, challenging conventional calibration practices reliant on homogeneous phantoms. The comparison protocol offers a framework for credentialing multi-institutional studies in FLASH preclinical research to enhance reproducibility of biologic findings., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Purpose: Ultrahigh-dose-rate (FLASH) irradiation has been reported to reduce normal tissue damage compared with conventional dose rate (CONV) irradiation without compromising tumor control. This proof-of-concept study aims to develop a deep learning (DL) approach to quantify the FLASH isoeffective dose (dose of CONV that would be required to produce the same effect as the given physical FLASH dose) with postirradiation mouse intestinal histology images., Methods and Materials: Eighty-four healthy C57BL/6J female mice underwent 16 MeV electron CONV (0.12 Gy/s; n = 41) or FLASH (200 Gy/s; n = 43) single fraction whole abdominal irradiation. Physical dose ranged from 12 to 16 Gy for FLASH and 11 to 15 Gy for CONV in 1 Gy increments. Four days after irradiation, 9 jejunum cross-sections from each mouse were hematoxylin and eosin stained and digitized for histological analysis. CONV data set was randomly split into training (n = 33) and testing (n = 8) data sets. ResNet101-based DL models were retrained using the CONV training data set to estimate the dose based on histological features. The classical manual crypt counting (CC) approach was implemented for model comparison. Cross-section-wise mean squared error was computed to evaluate the dose estimation accuracy of both approaches. The validated DL model was applied to the FLASH data set to map the physical FLASH dose into the isoeffective dose., Results: The DL model achieved a cross-section-wise mean squared error of 0.20 Gy 2 on the CONV testing data set compared with 0.40 Gy 2 of the CC approach. Isoeffective doses estimated by the DL model for FLASH doses of 12, 13, 14, 15, and 16 Gy were 12.19 ± 0.46, 12.54 ± 0.37, 12.69 ± 0.26, 12.84 ± 0.26, and 13.03 ± 0.28 Gy, respectively., Conclusions: Our proposed DL model achieved accurate CONV dose estimation. The DL model results indicate that in the physical dose range of 13 to 16 Gy, the biologic dose response of small intestinal tissue to FLASH irradiation is represented by a lower isoeffective dose compared with the physical dose. Our DL approach can be a tool for studying isoeffective doses of other radiation dose modifying interventions., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Current clinical diagnostic imaging methods for lung metastases are sensitive only to large tumours (1-2 mm cross-sectional diameter), and early detection can dramatically improve treatment. We have previously demonstrated that an antibody-targeted MRI contrast agent based on microparticles of iron oxide (MPIO; 1 μm diameter) enables the imaging of endothelial vascular cell adhesion molecule-1 (VCAM-1). Using a mouse model of lung metastasis, upregulation of endothelial VCAM-1 expression was demonstrated in micrometastasis-associated vessels but not in normal lung tissue, and binding of VCAM-MPIO to these vessels was evident histologically. Owing to the lack of proton MRI signals in the lungs, we modified the VCAM-MPIO to include zirconium-89 ( 89 Zr, t 1/2 = 78.4 h) in order to allow the in vivo detection of lung metastases by positron emission tomography (PET). Using this new agent ( 89 Zr-DFO-VCAM-MPIO), it was possible to detect the presence of micrometastases within the lung in vivo from ca. 140 μm in diameter. Histological analysis combined with autoradiography confirmed the specific binding of the agent to the VCAM-1 expressing vasculature at the sites of pulmonary micrometastases. By retaining the original VCAM-MPIO as the basis for this new molecular contrast agent, we have created a dual-modality (PET/MRI) agent for the concurrent detection of lung and brain micrometastases.

An immunosuppressive microenvironment causes poor tumor T cell infiltration and is associated with reduced patient overall survival in colorectal cancer. How to improve treatment responses in these tumors is still a challenge. Using an integrated screening approach to identify cancer-specific vulnerabilities, we identified complement receptor C5aR1 as a druggable target, which when inhibited improved radiotherapy, even in tumors displaying immunosuppressive features and poor CD8+ T cell infiltration. While C5aR1 is well-known for its role in the immune compartment, we found that C5aR1 is also robustly expressed on malignant epithelial cells, highlighting potential tumor cell-specific functions. C5aR1 targeting resulted in increased NF-κB-dependent apoptosis specifically in tumors and not normal tissues, indicating that, in malignant cells, C5aR1 primarily regulated cell fate. Collectively, these data revealed that increased complement gene expression is part of the stress response mounted by irradiated tumors and that targeting C5aR1 could improve radiotherapy, even in tumors displaying immunosuppressive features.

Background and Purpose: The impact of radiotherapy (RT) at ultra high vs conventional dose rate (FLASH vs CONV) on the generation and repair of DNA double strand breaks (DSBs) is an important question that remains to be investigated. Here, we tested the hypothesis as to whether FLASH-RT generates decreased chromosomal translocations compared to CONV-RT., Materials and Methods: We used two FLASH validated electron beams and high-throughput rejoin and genome-wide translocation sequencing (HTGTS-JoinT-seq), employing S. aureus and S. pyogenes Cas9 "bait" DNA double strand breaks (DSBs) in HEK239T cells, to measure differences in bait-proximal repair and their genome-wide translocations to "prey" DSBs generated after various irradiation doses, dose rates and oxygen tensions (normoxic, 21% O 2 ; physiological, 4% O 2 ; hypoxic, 2% and 0.5% O 2 ). Electron irradiation was delivered using a FLASH capable Varian Trilogy and the eRT6/Oriatron at CONV (0.08-0.13 Gy/s) and FLASH (1x10 2 -5x10 6 Gy/s) dose rates. Related experiments using clonogenic survival and γH2AX foci in the 293T and the U87 glioblastoma lines were also performed to discern FLASH-RT vs CONV-RT DSB effects., Results: Normoxic and physioxic irradiation of HEK293T cells increased translocations at the cost of decreasing bait-proximal repair but were indistinguishable between CONV-RT and FLASH-RT. Although no apparent increase in chromosome translocations was observed with hypoxia-induced apoptosis, the combined decrease in oxygen tension with IR dose-rate modulation did not reveal significant differences in the level of translocations nor in their junction structures. Furthermore, RT dose rate modality on U87 cells did not change γH2AX foci numbers at 1- and 24-hours post-irradiation nor did this affect 293T clonogenic survival., Conclusion: Irrespective of oxygen tension, FLASH-RT produces translocations and junction structures at levels and proportions that are indistinguishable from CONV-RT., Competing Interests: Declaration of Competing Interest BWL Jr. has received research support outside this work from Varian Medical Systems, is a co-founder and board member of TibaRay, and is a consultant on a clinical trial steering committee for BeiGene. PGM is a co-founder of TibaRay. The other authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Purpose: Ultrahigh-dose-rate (UHDR) radiation therapy (RT) has produced the FLASH effect in preclinical models: reduced toxicity with comparable tumor control compared with conventional-dose-rate RT. Early clinical trials focused on UHDR RT feasibility using specialized devices. We explore the technical feasibility of practical electron UHDR RT on a standard clinical linear accelerator (LINAC)., Methods and Materials: We tuned the program board of a decommissioned electron energy for UHDR electron delivery on a clinical LINAC without hardware modification. Pulse delivery was controlled using the respiratory gating interface. A short source-to-surface distance (SSD) electron setup with a standard scattering foil was configured and tested on an anthropomorphic phantom using circular blocks with 3- to 20-cm field sizes. Dosimetry was evaluated using radiochromic film and an ion chamber profiler., Results: UHDR open-field mean dose rates at 100, 80, 70, and 59 cm SSD were 36.82, 59.52, 82.01, and 112.83 Gy/s, respectively. At 80 cm SSD, mean dose rate was ∼60 Gy/s for all collimated field sizes, with an R80 depth of 6.1 cm corresponding to an energy of 17.5 MeV. Heterogeneity was <5.0% with asymmetry of 2.2% to 6.2%. The short SSD setup was feasible under realistic treatment conditions simulating broad clinical indications on an anthropomorphic phantom., Conclusions: Short SSD and tuning for high electron beam current on a standard clinical LINAC can deliver flat, homogenous UHDR electrons over a broad, clinically relevant range of field sizes and depths with practical working distances in a configuration easily reversible to standard clinical use., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Radiation therapy, one of the most effective therapies to treat cancer, is highly toxic to healthy tissue. The delivery of radiation at ultra-high dose rates, FLASH radiation therapy (FLASH), has been shown to maintain therapeutic anti-tumor efficacy while sparing normal tissues compared to conventional dose rate irradiation (CONV). Though promising, these studies have been limited mainly to murine models. Here, we leveraged enteroids, three-dimensional cell clusters that mimic the intestine, to study human-specific tissue response to radiation. We observed enteroids have a greater colony growth potential following FLASH compared with CONV. In addition, the enteroids that reformed following FLASH more frequently exhibited proper intestinal polarity. While we did not observe differences in enteroid damage across groups, we did see distinct transcriptomic changes. Specifically, the FLASH enteroids upregulated the expression of genes associated with the WNT-family, cell-cell adhesion, and hypoxia response. These studies validate human enteroids as a model to investigate FLASH and provide further evidence supporting clinical study of this therapy. Insight Box Promising work has been done to demonstrate the potential of ultra-high dose rate radiation (FLASH) to ablate cancerous tissue, while preserving healthy tissue. While encouraging, these findings have been primarily observed using pre-clinical murine and traditional two-dimensional cell culture. This study validates the use of human enteroids as a tool to investigate human-specific tissue response to FLASH. Specifically, the work described demonstrates the ability of enteroids to recapitulate previous in vivo findings, while also providing a lens through which to probe cellular and molecular-level responses to FLASH. The human enteroids described herein offer a powerful model that can be used to probe the underlying mechanisms of FLASH in future studies., (© The Author(s) 2023. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

The rapid depletion of oxygen during irradiation at ultra-high dose rate calls for tissue oximeters capable of high temporal resolution. This study demonstrates a water-soluble phosphorescent nanoprobe and fiber-coupled instrument, which together are used to measure the kinetics of oxygen depletion at 200 Hz during irradiation of in vitro solutions., Competing Interests: Declaration of competing interest BWL has received research support from Varian Medical Systems. BWL is a co-founder and board member of TibaRay. Other authors declare that they have no competing interests., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Rationale: The role of neutrophils and their extracellular vesicles (EVs) in the pathogenesis of pulmonary arterial hypertension is unclear. Objectives: To relate functional abnormalities in pulmonary arterial hypertension neutrophils and their EVs to mechanisms uncovered by proteomic and transcriptomic profiling. Methods: Production of elastase, release of extracellular traps, adhesion, and migration were assessed in neutrophils from patients with pulmonary arterial hypertension and control subjects. Proteomic analyses were applied to explain functional perturbations, and transcriptomic data were used to find underlying mechanisms. CD66b-specific neutrophil EVs were isolated from plasma of patients with pulmonary arterial hypertension, and we determined whether they produce pulmonary hypertension in mice. Measurements and Main Results: Neutrophils from patients with pulmonary arterial hypertension produce and release increased neutrophil elastase, associated with enhanced extracellular traps. They exhibit reduced migration and increased adhesion attributed to elevated β1-integrin and vinculin identified by proteomic analysis and previously linked to an antiviral response. This was substantiated by a transcriptomic IFN signature that we related to an increase in human endogenous retrovirus K envelope protein. Transfection of human endogenous retrovirus K envelope in a neutrophil cell line (HL-60) increases neutrophil elastase and IFN genes, whereas vinculin is increased by human endogenous retrovirus K deoxyuridine triphosphate diphosphatase that is elevated in patient plasma. Neutrophil EVs from patient plasma contain increased neutrophil elastase and human endogenous retrovirus K envelope and induce pulmonary hypertension in mice, mitigated by elafin, an elastase inhibitor. Conclusions: Elevated human endogenous retroviral elements and elastase link a neutrophil innate immune response to pulmonary arterial hypertension.

Background and Purpose: We describe a multicenter cross validation of ultra-high dose rate (UHDR) (>= 40 Gy/s) irradiation in order to bring a dosimetric consensus in absorbed dose to water. UHDR refers to dose rates over 100-1000 times those of conventional clinical beams. UHDR irradiations have been a topic of intense investigation as they have been reported to induce the FLASH effect in which normal tissues exhibit reduced toxicity relative to conventional dose rates. The need to establish optimal beam parameters capable of achieving the in vivo FLASH effect has become paramount. It is therefore necessary to validate and replicate dosimetry across multiple sites conducting UHDR studies with distinct beam configurations and experimental set-ups., Materials and Methods: Using a custom cuboid phantom with a cylindrical cavity (5 mm diameter by 10.4 mm length) designed to contain three type of dosimeters (thermoluminescent dosimeters (TLDs), alanine pellets, and Gafchromic films), irradiations were conducted at expected doses of 7.5 to 16 Gy delivered at UHDR or conventional dose rates using various electron beams at the Radiation Oncology Departments of the CHUV in Lausanne, Switzerland and Stanford University, CA., Results: Data obtained between replicate experiments for all dosimeters were in excellent agreement (±3%). In general, films and TLDs were in closer agreement with each other, while alanine provided the closest match between the expected and measured dose, with certain caveats related to absolute reference dose., Conclusion: In conclusion, successful cross-validation of different electron beams operating under different energies and configurations lays the foundation for establishing dosimetric consensus for UHDR irradiation studies, and, if widely implemented, decrease uncertainty between different sites investigating the mechanistic basis of the FLASH effect., (Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.)

Treatment of advanced ovarian cancer using PD-1/PD-L1 immune checkpoint blockade shows promise; however, current clinical trials are limited by modest response rates. Radiotherapy has been shown to synergize with PD-1/PD-L1 blockade in some cancers but has not been utilized in advanced ovarian cancer due to toxicity associated with conventional abdominopelvic irradiation. Ultrahigh-dose rate (FLASH) irradiation has emerged as a strategy to reduce radiation-induced toxicity, however, the immunomodulatory properties of FLASH irradiation remain unknown. Here, we demonstrate that single high-dose abdominopelvic FLASH irradiation promoted intestinal regeneration and maintained tumor control in a preclinical mouse model of ovarian cancer. Reduced tumor burden in conventional and FLASH-treated mice was associated with an early decrease in intratumoral regulatory T cells and a late increase in cytolytic CD8 + T cells. Compared with conventional irradiation, FLASH irradiation increased intratumoral T-cell infiltration at early timepoints. Moreover, FLASH irradiation maintained the ability to increase intratumoral CD8 + T-cell infiltration and enhance the efficacy of αPD-1 therapy in preclinical models of ovarian cancer. These data highlight the potential for FLASH irradiation to improve the therapeutic efficacy of checkpoint inhibition in the treatment of ovarian cancer., (©2021 American Association for Cancer Research.)

Purpose: The differential response of normal and tumor tissues to ultrahigh-dose-rate radiation (FLASH) has raised new hope for treating solid tumors but, to date, the mechanism remains elusive. One leading hypothesis is that FLASH radiochemically depletes oxygen from irradiated tissues faster than it is replenished through diffusion. The purpose of this study was to investigate these effects within hypoxic multicellular tumor spheroids through simulations and experiments., Methods and Materials: Physicobiological equations were derived to model (1) the diffusion and metabolism of oxygen within spheroids; (2) its depletion through reactions involving radiation-induced radicals; and (3) the increase in radioresistance of spheroids, modeled according to the classical oxygen enhancement ratio and linear-quadratic response. These predictions were then tested experimentally in A549 spheroids exposed to electron irradiation at conventional (0.075 Gy/s) or FLASH (90 Gy/s) dose rates. Clonogenic survival, cell viability, and spheroid growth were scored postradiation. Clonogenic survival of 2 other cell lines was also investigated., Results: The existence of a hypoxic core in unirradiated tumor spheroids is predicted by simulations and visualized by fluorescence microscopy. Upon FLASH irradiation, this hypoxic core transiently expands, engulfing a large number of well-oxygenated cells. In contrast, oxygen is steadily replenished during slower conventional irradiation. Experimentally, clonogenic survival was around 3-fold higher in FLASH-irradiated spheroids compared with conventional irradiation, but no significant difference was observed for well-oxygenated 2-dimensional cultured cells. This differential survival is consistent with the predictions of the computational model. FLASH irradiation of spheroids resulted in a dose-modifying factor of around 1.3 for doses above 10 Gy., Conclusions: Tumor spheroids can be used as a model to study FLASH irradiation in vitro. The improved survival of tumor spheroids receiving FLASH radiation confirms that ultrafast radiochemical oxygen depletion and its slow replenishment are critical components of the FLASH effect., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Radiation therapy is the most effective cytotoxic therapy for localized tumors. However, normal tissue toxicity limits the radiation dose and the curative potential of radiation therapy when treating larger target volumes. In particular, the highly radiosensitive intestine limits the use of radiation for patients with intra-abdominal tumors. In metastatic ovarian cancer, total abdominal irradiation (TAI) was used as an effective postsurgical adjuvant therapy in the management of abdominal metastases. However, TAI fell out of favor due to high toxicity of the intestine. Here we utilized an innovative preclinical irradiation platform to compare the safety and efficacy of TAI ultra-high dose rate FLASH irradiation to conventional dose rate (CONV) irradiation in mice. We demonstrate that single high dose TAI-FLASH produced less mortality from gastrointestinal syndrome, spared gut function and epithelial integrity, and spared cell death in crypt base columnar cells compared to TAI-CONV irradiation. Importantly, TAI-FLASH and TAI-CONV irradiation had similar efficacy in reducing tumor burden while improving intestinal function in a preclinical model of ovarian cancer metastasis. These findings suggest that FLASH irradiation may be an effective strategy to enhance the therapeutic index of abdominal radiotherapy, with potential application to metastatic ovarian cancer.

Radiation therapy, along with surgery and chemotherapy, is one of the main treatments for cancer. While radiotherapy is highly effective in the treatment of localized tumors, its main limitation is its toxicity to normal tissue. Previous preclinical studies have reported that ultra-high dose-rate (FLASH) irradiation results in reduced toxicity to normal tissues while controlling tumor growth to a similar extent relative to conventional-dose-rate (CONV) irradiation. To our knowledge this is the first report of a dose-response study in mice comparing the effect of FLASH irradiation vs. CONV irradiation on skin toxicity. We found that FLASH irradiation results in both a lower incidence and lower severity of skin ulceration than CONV irradiation 8 weeks after single-fraction hemithoracic irradiation at high doses (30 and 40 Gy). Survival was also higher after FLASH hemithoracic irradiation (median survival >180 days at doses of 30 and 40 Gy) compared to CONV irradiation (median survival 100 and 52 days at 30 and 40 Gy, respectively). No ulceration was observed at doses 20 Gy or below in either FLASH or CONV. These results suggest a shifting of the dose-response curve for radiation-induced skin ulceration to the right for FLASH, compared to CONV irradiation, suggesting the potential for an enhanced therapeutic index for radiation therapy of cancer., (©2020 by Radiation Research Society. All rights of reproduction in any form reserved.)

The observation of an enhanced therapeutic index for FLASH radiotherapy in mice has created interest in practical laboratory-based FLASH irradiators. To date, systems capable of 3D conformal FLASH irradiation in mice have been lacking. We are developing such a system, incorporating a high-current linear accelerator to produce a collimated X-ray beam in a stationary beamline design, rotating the mouse about a longitudinal axis to achieve conformal irradiation from multiple beam directions. The purpose of this work was to evaluate the reproducibility of mouse anatomy under rotation at speeds compatible with conformal FLASH delivery. Three short-hair mice and two hairless mice were immobilized under anesthesia in body weight-specific contoured plastic molds, and subjected to three rotational (up to 3 revolutions/s) and two non-rotational movement interventions. MicroCT images were acquired before and after each intervention. The displacements of 11 anatomic landmarks were measured on the image pairs. The displacement of the anatomical landmarks with any of the interventions was 0.5 mm or less for 92.4% of measurements, with a single measurement out of 275 (11 landmarks × 5 interventions × 5 mice) reaching 1 mm. There was no significant difference in the displacements associated with rotation compared to those associated with moving the immobilized mouse in and out of a scanner or with leaving the mouse in place for 5 min with no motion. There were no significant differences in displacements between mice with or without hair, although the analysis is limited by small numbers, or between different anatomic landmarks. These results show that anatomic reproducibility under rotation speed corresponding to FLASH irradiation times appears to be compatible with conformal/stereotactic irradiation in mice., (©2020 by Radiation Research Society. All rights of reproduction in any form reserved.)

The efficacy of dose-enhancing gold nanoparticles (AuNPs) is negatively impacted by low tumor uptake, low cell membrane penetration, limited diffusion distance, and short lifetime of radiation-induced secondary particles. To overcome these limitations, we have developed a novel AuNP system capable of radiation-triggered release of nitrite, a precursor of reactive nitrogen species, and report here on the in vivo characterization of this system. AuNPs were functionalized through PEGylation, cell-penetrating peptides (CPP; AuNP@CPP), and nitroimidazole (nIm; AuNP@nIm-CPP). Mice with subcutaneous 4T1 tumors received either AuNP@nIm-CPP or AuNP@CPP intraperitoneally. Tumor and normal tissue uptake were evaluated 24 h post AuNP administration. A separate cohort of mice was injected and irradiated to a single-fraction dose of 18 Gy in a 225 kVp small animal irradiator 24 h post NP administration. The mice were followed for two weeks to evaluate tumor response. The mean physical and hydrodynamic size of both NP systems were 5 and 13 nm, respectively. NP nIm-loading of 1 wt% was determined. Tumor accumulation of AuNP@nIm-CPP was significantly lower than that of AuNP@CPP (0.2% vs 1.2%, respectively). In contrast, AuNP@nIm-CPP showed higher accumulation compared to AuNP@CPP in liver (16.5% vs 6.6%, respectively) and spleen (10.8% vs 3.1%, respectively). With respect to tumor response, no differential response was found between non-irradiated mice receiving either saline or AuNP@nIm-CPP alone. The combination of AuNP@CPP+ radiation showed no differential response from radiation alone. In contrast, a significant delay in tumor regrowth was observed in mice receiving AuNP@nIm-CPP+ radiation compared to radiation alone. AuNP functionalized with both CPP and nIm exhibited an order of magnitude less tumor accumulation compared to the NP system without nIm yet resulted in a significantly higher therapeutic response. Our data suggest that by improving the biokinetics of AuNP@nIm-CPP, this novel NP system could be a promising radiosensitizer for enhanced therapeutic response following radiation therapy.

Breast cancer is the most common cancer among American women and a major cause of mortality. To identify metabolic pathways as potential targets to treat metastatic breast cancer, we performed metabolomics profiling on the breast cancer cell line MDA-MB-231 and its tissue-tropic metastatic subclones. Here, we report that these subclones with increased metastatic potential display an altered metabolic profile compared with the parental population. In particular, the mitochondrial serine and one-carbon (1C) unit pathway is upregulated in metastatic subclones. Mechanistically, the mitochondrial serine and 1C unit pathway drives the faster proliferation of subclones through enhanced de novo purine biosynthesis. Inhibition of the first rate-limiting enzyme of the mitochondrial serine and 1C unit pathway, serine hydroxymethyltransferase (SHMT2), potently suppresses proliferation of metastatic subclones in culture and impairs growth of lung metastatic subclones at both primary and metastatic sites in mice. Some human breast cancers exhibit a significant association between the expression of genes in the mitochondrial serine and 1C unit pathway with disease outcome and higher expression of SHMT2 in metastatic tumor tissue compared with primary tumors. In addition to breast cancer, a few other cancer types, such as adrenocortical carcinoma and kidney chromophobe cell carcinoma, also display increased SHMT2 expression during disease progression. Together, these results suggest that mitochondrial serine and 1C unit metabolism plays an important role in promoting cancer progression, particularly in late-stage cancer. IMPLICATIONS: This study identifies mitochondrial serine and 1C unit metabolism as an important pathway during the progression of a subset of human breast cancers., (©2020 American Association for Cancer Research.)

Despite considerable progress with our understanding of glioblastoma multiforme (GBM) and the precise delivery of radiotherapy, the prognosis for GBM patients is still unfavorable with tumor recurrence due to radioresistance being a major concern. We recently developed a cross-linked iron oxide nanoparticle conjugated to azademethylcolchicine (CLIO-ICT) to target and eradicate a subpopulation of quiescent cells, glioblastoma initiating cells (GICs), which could be a reason for radioresistance and tumor relapse. The purpose of our study was to investigate if CLIO-ICT has an additive therapeutic effect to enhance the response of GBMs to ionizing radiation. Methods: NSG™ mice bearing human GBMs and C57BL/6J mice bearing murine GBMs received CLIO-ICT, radiation, or combination treatment. The mice underwent pre- and post-treatment magnetic resonance imaging (MRI) scans, bioluminescence imaging (BLI), and histological analysis. Tumor nanoparticle enhancement, tumor flux, microvessel density, GIC, and apoptosis markers were compared between different groups using a one-way ANOVA and two-tailed Mann-Whitney test. Additional NSG™ mice underwent survival analyses with Kaplan-Meier curves and a log rank (Mantel-Cox) test. Results: At 2 weeks post-treatment, BLI and MRI scans revealed significant reduction in tumor size for CLIO-ICT plus radiation treated tumors compared to monotherapy or vehicle-treated tumors. Combining CLIO-ICT with radiation therapy significantly decreased microvessel density, decreased GICs, increased caspase-3 expression, and prolonged the survival of GBM-bearing mice. CLIO-ICT delivery to GBM could be monitored with MRI. and was not significantly different before and after radiation. There was no significant caspase-3 expression in normal brain at therapeutic doses of CLIO-ICT administered. Conclusion: Our data shows additive anti-tumor effects of CLIO-ICT nanoparticles in combination with radiotherapy. The combination therapy proposed here could potentially be a clinically translatable strategy for treating GBMs., Competing Interests: Competing Interests: HEDL, RAF, and PML hold a joint patent on the described theranostic nanoparticles (WO 2015014756)., (© The author(s).)

The complement system is an innate immune pathway typically thought of as part of the first line of defence against "non-self" species. In the context of cancer, complement has been described to have an active role in facilitating cancer-associated processes such as increased proliferation, angiogenesis and migration. Several cellular members of the tumour microenvironment express and/or produce complement proteins locally, including tumour cells. Dysregulation of the complement system has been reported in numerous tumours and increased expression of complement activation fragments in cancer patient specimens correlates with poor patient prognosis. Importantly, genetic or pharmacological targeting of complement has been shown to reduce tumour growth in several cancer preclinical models, suggesting that complement could be an attractive therapeutic target. Hypoxia (low oxygen) is frequently found in solid tumours and has a profound biological impact on cellular and non-cellular components of the tumour microenvironment. In this review, we focus on hypoxia since this is a prevailing feature of the tumour microenvironment that, like increased complement, is typically associated with poor prognosis. Furthermore, interesting links between hypoxia and complement have been recently proposed but never collectively reviewed. Here, we explore how hypoxia alters regulation of complement proteins in different cellular components of the tumour microenvironment, as well as the downstream biological consequences of this regulation.

Although radiotherapy (RT) decreases the incidence of locoregional recurrence in breast cancer, patients with triple-negative breast cancer (TNBC) have increased risk of local recurrence following breast-conserving therapy. The relationship between RT and local recurrence is unknown. Here, we tested the hypothesis that recurrence in some instances is due to the attraction of circulating tumor cells to irradiated tissues. To evaluate the effect of absolute lymphocyte count on local recurrence after RT in patients with TNBC, we analyzed radiation effects on tumor and immune cell recruitment to tissues in an orthotopic breast cancer model. Recurrent patients exhibited a prolonged low absolute lymphocyte count when compared with nonrecurrent patients following RT. Recruitment of tumor cells to irradiated normal tissues was enhanced in the absence of CD8 + T cells. Macrophages (CD11b + F480 + ) preceded tumor cell infiltration and were recruited to tissues following RT. Tumor cell recruitment was mitigated by inhibiting macrophage infiltration using maraviroc, an FDA-approved CCR5 receptor antagonist. Our work poses the intriguing possibility that excessive macrophage infiltration in the absence of lymphocytes promotes local recurrence after RT. This combination thus defines a high-risk group of patients with TNBC. Significance: This study establishes the importance of macrophages in driving tumor cell recruitment to sites of local radiation therapy and suggests that this mechanism contributes to local recurrence in women with TNBC that are also immunosuppressed. Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/78/15/4241/F1.large.jpg Cancer Res; 78(15); 4241-52. ©2018 AACR ., (©2018 American Association for Cancer Research.)

Angiogenesis is an essential component of tumour growth and, consequently, an important target both therapeutically and diagnostically. The cell adhesion molecule α(v)β(3) integrin is a specific marker of angiogenic vessels and the most prevalent vascular integrin that binds the amino acid sequence arginine-glycine-aspartic acid (RGD). Previous studies using RGD-targeted nanoparticles (20-50 nm diameter) of iron oxide (NPIO) for magnetic resonance imaging (MRI) of tumour angiogenesis, have identified a number of limitations, including non-specific extravasation, long blood half-life (reducing specific contrast) and low targeting valency. The aim of this study, therefore, was to determine whether conjugation of a cyclic RGD variant [c(RGDyK)], with enhanced affinity for α(v)β(3), to microparticles of iron oxide (MPIO) would provide a more sensitive contrast agent for imaging of angiogenic tumour vessels. Cyclic RGD [c(RGDyK)] and RAD [c(RADyK)] based peptides were coupled to 2.8 μm MPIO, and binding efficacy tested both in vitro and in vivo. Significantly greater specific binding of c(RGDyK)-MPIO to S-nitroso-n-acetylpenicillamine (SNAP)-stimulated human umbilical vein endothelial cells in vitro than PBS-treated cells was demonstrated under both static (14-fold increase; P < 0.001) and flow (44-fold increase; P < 0.001) conditions. Subsequently, mice bearing subcutaneous colorectal (MC38) or melanoma (B16F10) derived tumours underwent in vivo MRI pre- and post-intravenous administration of c(RGDyK)-MPIO or c(RADyK)-MPIO. A significantly greater volume of MPIO-induced hypointensities were found in c(RGDyK)-MPIO injected compared to c(RADyK)-MPIO injected mice, in both tumour models (P < 0.05). Similarly, administration of c(RGDyK)-MPIO induced a greater reduction in mean tumour T(2)* relaxation times than the control agent in both tumour models (melanoma P < 0.001; colorectal P < 0.0001). Correspondingly, MPIO density per tumour volume assessed immunohistochemically was significantly greater for c(RGDyK)-MPIO than c(RADyK)-MPIO injected animals, in both melanoma (P < 0.05) and colorectal (P < 0.0005) tumours. In both cases, binding of c(RGDyK)-MPIO co-localised with α(v)β(3) expression. Comparison of RGD-targeted and dynamic contrast enhanced (DCE) MRI assessment of tumour perfusion indicated sensitivity to different vascular features. This study demonstrates specific binding of c(RGDyK)-MPIO to α(v)β(3) expressing neo-vessels, with marked and quantifiable contrast and rapid clearance of unbound particles from the blood circulation compared to NPIO. Combination of this molecular MRI approach with conventional DCE MRI will enable integrated molecular, anatomical and perfusion tumour imaging.