Small Animal Radiation Research Research Articles

Targeted irradiation in an autochthonous mouse model of pancreatic cancer.

The value of radiotherapy in the treatment of pancreatic cancer has been the subject of much debate but limited preclinical research. We hypothesise that the poor translation of radiation research into clinical trials of radiotherapy in pancreatic cancer is due, in part, to inadequate preclinical study models. Here, we developed and refined methods for targeted irradiation in autochthonous mouse models of pancreatic cancer, using a small animal radiotherapy research platform. We tested and optimised strategies for administration of contrast agents, iohexol and the liver imaging agent Fenestra LC, to enable the use of computed tomography imaging in tumour localisation. We demonstrate accurate tumour targeting, negligible off-target effects and therapeutic efficacy, depending on dose, number of fractions and tumour size, and provide a proof of concept that precise radiation can be delivered effectively to mouse pancreatic tumours with a clinically relevant microenvironment. This advance will allow investigation of the radiation response in murine pancreatic cancer, discovery of mechanisms and biomarkers of radiosensitivity or resistance, and development of radiosensitising strategies to inform clinical trials for precision radiotherapy in this disease.

A multimodality assessment of the protective capacity of statin therapy in a mouse model of radiation cardiotoxicity

PurposeDespite technological advances in radiotherapy (RT), cardiotoxicity remains a common complication in patients with lung, oesophageal and breast cancers. Statin therapy has been shown to have pleiotropic properties beyond its lipid-lowering effects. Previous murine models have shown statin therapy can reduce short-term functional effects of whole-heart irradiation. In this study, we assessed the efficacy of atorvastatin in protecting against the late effects of radiation exposure on systolic function, cardiac conduction, and atrial natriuretic peptide (ANP) following a clinically relevant partial-heart radiation exposure. Materials and MethodsFemale, 12-week old, C57BL/6j mice received an image-guided 16 Gy X-ray field to the base of the heart using a small animal radiotherapy research platform (SARRP), with or without atorvastatin from 1 week prior to irradiation until the end of the experiment. The animals were followed for 50 weeks with longitudinal transthoracic echocardiography (TTE) and electrocardiography (ECG) every 10 weeks, and plasma ANP every 20 weeks. ResultsAt 30–50 weeks, mild left ventricular systolic function impairment observed in the RT control group was less apparent in animals receiving atorvastatin. ECG analysis demonstrated prolongation of components of cardiac conduction related to the heart base at 10 and 30 weeks in the RT control group but not in animals treated with atorvastatin. In contrast to systolic function, conduction disturbances resolved at later time-points with radiation alone. ANP reductions were lower in irradiated animals receiving atorvastatin at 30 and 50 weeks. ConclusionsAtorvastatin prevents left ventricular systolic dysfunction, and the perturbation of cardiac conduction following partial heart irradiation. If confirmed in clinical studies, these data would support the use of statin therapy for cardioprotection during thoracic radiotherapy.

Cardiac sub-volume targeting demonstrates regional radiosensitivity in the mouse heart

Background and purposeRadiation-induced cardiac toxicity (RICT) remains one of the most critical dose limiting constraints in radiotherapy. Recent studies have shown higher doses to the base of the heart are associated with worse overall survival in lung cancer patients receiving radiotherapy. This work aimed to investigate the impact of sub-volume heart irradiation in a mouse model using small animal image-guided radiotherapy. Materials and methodsC57BL/6 mice were irradiated with a single fraction of 16 Gy to the base, middle or apex of the heart using a small animal radiotherapy research platform. Cone beam CT and echocardiography were performed at baseline and at 10 week intervals until 50 weeks post-treatment. Structural and functional parameters were correlated with mean heart dose (MHD) and volume of heart receiving 5 Gy (V5). ResultsAll irradiated mice showed a time dependent increase in left ventricle wall thickness in diastole of ~0.2 mm detected at 10 weeks post-treatment, with the most significant and persistent changes occurring in the heart base-irradiated animals. Similarly, statistically different functional effects (p < 0.01) were observed in base-irradiated animals which showed the most significant decreases compared to controls. The observed functional changes did not correlate with MHD and V5 (R2 < 0.1), indicating that whole heart dosimetry parameters do not predict physiological changes resulting from cardiac sub-volume irradiation. ConclusionsThis is the first report demonstrating the structural and functional consequences of sub-volume targeting in the mouse heart and reverse translates clinical observations indicating the heart base as a critical radiosensitive region.

Development of a Compact PET for integrated PET/CT/RT to Streamline and Enhance Functional/Anatomic Image-Guided Preclinical Radiation Oncology Researches

To present latest progress in developing a compact and high-performance PET that will be inserted inside an existing CT image-guided small animal irradiator to establish an integrated PET/CT/RT for substantially expanded and improved image-guided RT research capabilities. 1) PET will consist of 12 detector panels in a multi-polygon configuration to provide ∼10 cm diameter trans-axial and ∼3.2 cm axial image field-of-view: Each panel consists of 4 detectors that closely tiled in a 2x2 matrix: Each detector consists of 15x15 array of 1x1x20 mm LYSO scintillators, with each end of the scintillator array read out by an 8x8 array of 2x2 mm Silicon Photomultiplier (SiPM) array to provide dual-scintillator-end readout for measuring depth-of-interaction (DOI); 2) PC Board of discrete components based electronics have been developed for reading/processing total 768 detector output channels (after readout channel reduction) with both positive and negative polarity signals from SiPMs’ anodes and cathodes; 3) Standard detector performance evaluation have been conducted, including energy, coincidence timing, DOI, and intrinsic detector spatial resolutions; 4) Imaging performance based on rotating detector panels is under investigation. Two full detector panels have already been assembled and placed opposite to each other on a rotating table, with a radioactive rod phantom between the panels. Coincident data will be acquired by the detector pairs at 6 different rotating angles span over 180 degrees, with 30 degrees between each consecutive detector angles. Data corrections, including attenuation, scatter and random will be applied before the image reconstruction. Expanded dual positive and negative polarity signal readout and processing electronics works well. All 900 crystals on each detector panel can be very well identified; energy, coincidence timing, and DOI are 18%-34%, ∼2.0 ns, and 2.4 – 3.7 mm, respectively. The detector intrinsic spatial resolution is < 1.0 mm. Overall, detectors have achieved expected performance. As anticipated, ongoing phantom imaging study with dual rotating detector panels, potentially along with the performance evaluation study of the assembled PET system, will be reported in the conference. We have developed PET detectors and readout/processing electronics with desired performance, and expect the ongoing system construction and imaging studies will quantify the PET capability for functional image guidance for small animal radiotherapy research.

Towards a novel small animal proton irradiation platform: the SIRMIO project

Background: Precision small animal radiotherapy research is a young emerging field aiming to provide new experimental insights into tumor and normal tissue models in different microenvironments, to unravel complex mechanisms of radiation damage in target and non-target tissues and assess efficacy of novel therapeutic strategies. For photon therapy, modern small animal radiotherapy research platforms have been developed over the last years and are meanwhile commercially available. Conversely, for proton therapy, which holds potential for an even superior outcome than photon therapy, no commercial system exists yet.Material and methods: The project SIRMIO (Small Animal Proton Irradiator for Research in Molecular Image-guided Radiation-Oncology) aims at realizing and demonstrating an innovative portable prototype system for precision image-guided small animal proton irradiation, suitable for installation at existing clinical treatment facilities. The proposed design combines precise dose application with in situ multi-modal anatomical image guidance and in vivo verification of the actual treatment delivery.Results and conclusions: This manuscript describes the status of the different components under development, featuring a dedicated beamline for degradation and focusing of clinical proton beams, along with novel detector systems for in situimaging and range verification. The foreseen workflow includes pre-treatment proton transmission imaging, complemented by ultrasonic tumor localization, for treatment planning and position verification, followed by image-guided delivery with on site range verification by means of ionoacoustics (for pulsed beams) and positron-emission-tomography (PET, for continuous beams). The proposed compact and cost-effective system promises to open a new era in small animal proton therapy research, contributing to the basic understanding of in vivo radiation action to identify areas of potential breakthroughs for future translation into innovative clinical strategies.

Development of an anatomically correct mouse phantom for dosimetry measurement in small animal radiotherapy research

Significant improvements in radiotherapy are likely to come from biological rather than technical optimization, for example increasing tumour radiosensitivity via combination with targeted therapies. Such paradigms must first be evaluated in preclinical models for efficacy, and recent advances in small animal radiotherapy research platforms allow advanced irradiation protocols, similar to those used clinically, to be carried out in orthotopic models. Dose assessment in such systems is complex however, and a lack of established tools and methodologies for traceable and accurate dosimetry is currently limiting the capabilities of such platforms and slowing the clinical uptake of new approaches. Here we report the creation of an anatomically correct phantom, fabricated from materials with tissue-equivalent electron density, into which dosimetry detectors can be incorporated for measurement as part of quality control (QC). The phantom also allows training in preclinical radiotherapy planning and cross-institution validation of dose delivery protocols for small animal radiotherapy platforms without the need to sacrifice animals, with high reproducibility.Mouse CT data was acquired and segmented into soft tissue, bone and lung. The skeleton was fabricated using 3D printing, whilst lung was created using computer numerical control (CNC) milling. Skeleton and lung were then set into a surface-rendered mould and soft tissue material added to create a whole-body phantom. Materials for fabrication were characterized for atomic composition and attenuation for x-ray energies typically found in small animal irradiators. Finally cores were CNC milled to allow intracranial incorporation of bespoke detectors (alanine pellets) for dosimetry measurement.

Mild hyperthermia as a localized radiosensitizer for deep-seated tumors: investigation in an orthotopic prostate cancer model in mice.

Non-ablative or mild hyperthermia (HT) has been shown in preclinical (and clinical) studies as a localized radiosensitizer that enhances the tumoricidal effects of radiation. Most preclinical in vivo HT studies use subcutaneous tumor models which do not adequately represent clinical conditions (e.g. proximity of normal/critical organs) or replicate the tumor microenvironment-both of which are important factors for eventual clinical translation. The purpose of this work is to demonstrate proof-of-concept of locoregional radiosensitization with superficially applied, radiofrequency (RF)-induced HT in an orthotopic mouse model of prostate cancer. In a 4-arm study, 40 athymic male nude mice were inoculated in the prostate with luciferase-transfected human prostate cancer cells (PC3). Tumor volumes were allowed to reach 150-250 mm3 (as measured by ultrasound) following which, mice were randomized into (i) control (no intervention); (ii) HT alone; (iii) RT alone; and (iv) HT + RT. RF-induced HT was administered (Groups ii and iv) using the Oncotherm LAB EHY-100 device to achieve a target temperature of 41 °C in the prostate. RT was administered ~30 min following HT, using an image-guided small animal radiotherapy research platform. In each case, a dual arc plan was used to deliver 12 Gy to the target in a single fraction. One animal from each cohort was euthanized on Day 10 or 11 after treatment for caspase-9 and caspase-3 Western blot analysis. The inoculation success rate was 89%. Mean tumor size at randomization (~16 days post-inoculation) was ~189 mm3 . Following the administration of RT and HT, mean tumor doubling times in days were: control = 4.2; HT = 4.5; RT = 30.4; and HT + RT = 33.4. A significant difference (p = 0.036) was noted between normalized nadir volumes for the RT alone (0.76) and the HT + RT (0.40) groups. Increased caspase-3 expression was seen in the combination treatment group compared to the other treatment groups. These early results demonstrate the successful use of external mild HT as a localized radiosensitizer for deep-seated tumors. We successfully demonstrated the feasibility of administering external mild HT in an orthotopic tumor model and demonstrated preclinical proof-of-concept of HT-based localized radiosensitization in prostate cancer radiotherapy.

Evaluation of four different small animal radiation plans on tumour and normal tissue dosimetry in a glioblastoma mouse model.

Small animal radiotherapy research platforms such as XStrahl's SARRP enable more precise irradiation of tumours and normal tissues in pre-clinical models of cancer. Using an orthotopic G7 glioblastoma xenograft model we studied the impact of four different radiotherapy plans on tumour and normal tissue dosimetry. Plans were created using four different approaches (single beam, parallel opposed pair, single plane arcs, couch rotation arcs) and dose volume histograms (DVH) for the tumour and the relevant organs at risk (OARs) (mouth, ipsilateral brain, contralateral brain, brain stem) were compared for a sample mouse subject. To evaluate the accuracy of delivery, treatment plans were recreated in solid-water phantoms and delivered to radiochromic film. Favourable tumour dosimetry was achieved by all plans. DVH analysis showed that different plans could be used to spare specific OARs depending on the objectives of the study. The delivery accuracy of the various treatments was better than 2%/2mm (dose difference/distance to agreement) in terms of global γ analysis. Small animal radiotherapy research platforms are an exciting addition to the pre-clinical research environment. Such systems improve the conformality of irradiation of tumours and OARs while maintaining a high degree of accuracy and enable investigators to optimise experiments in terms of tumour coverage and inclusion or exclusion of relevant OARs. This study confirms the utility of the SARRP in terms of the accuracy of plan delivery, and informs decisions on treatment planning to optimise the clinical relevance and scientific value of experiments.

Quality Assurance of Small Animal Irradiation: Validation of a 3D-Printed Phantom for “Quasi In-Vivo” Dosimetry

New therapeutic options in radiation oncology are often explored in preclinical in vivo studies using small animals. In this study we report on the use of a 3D-printed rat phantom for radiotherapy dosimetric “quasi-in vivo”quality assurance. In the context of a research project, we aimed to irradiate with a dose of 2Gy per fraction the half-brain of a rat model as homogeneously as possible and with maximal sparing of the contralateral half-brain. The dose was delivered by a 4 MV X-ray beam using a direct anterior-posterior, hemi-field beam, with a size of 3cm x 4cm. A 2cm thick 3D-printed bolus made of poly lactic acid (PLA) was used to avoid the build-up region of the beam in the target volume. Target alignment within the planned position was performed using online kilo-voltage on-board imaging. To verify proper dose delivery, considering the small size of the animal and the region to treat, a 3D 1:1 scale printed copy of the rat body was produced from the CT dataset of the rat to be used as a phantom for dose measurements. A plane section of the phantom perpendicular to the beam axis was created to insert thermoluminescent dosimeters (TLDs) and accommodate a radiochromic film. TLD measurements were made in field and out of field in the brain region, and just beneath the bolus, and were repeated 3 times per position. Kilo-voltage doses as well as linac daily output dose fluctuations were taken into account for dose corrections. The radiochromic film measurements were performed to analyse the dose profiles within the brain, from the cold to the hot region. Radiochromic film dosimetry showed a good concordance between the planned dose distribution and the measured dose, with the steep dose gradient at the edge of the hemi-field properly aligned to spare the contralateral half-brain. Mean TLDs percentage dose differences, (measurements minus planned/ planned), were 1.3% in-field and 0.9% beneath bolus. Out of field, dose measurements gave a mean of 0.05Gy for an expected dose of 0.04Gy. A 3D-printed 1:1 scale rat phantom is an effective and reliable tool for “quasi in-vivo” dosimetric quality assurance in the setting of small animal radiotherapy research.

Abstract 4153: Commissioning and radiobiological verification of the small animal radiotherapy research platform (SARRP)

Abstract Preclinical radiation biology has become increasingly sophisticated due to the implementation of advanced small animal image guided radiation platforms into laboratory investigation. Small animal radiotherapy devices enable state-of-the-art image guided therapy (IGRT) research to be performed in small animal cancer models by combining high-resolution cone beam computed tomography (CBCT) imaging with an isocentric irradiation system. The technological evolution from simple, broad field irradiation configurations, to more sophisticated dose deliveries for preclinical radiobiology experiments has introduced new dosimetry challenges for preclinical small animal cancer research. Because of this, robust QA and dosimetry techniques are a key part of using novel treatment platforms using very small irradiation fields. In this study, we present a dosimetric study of the small animal radiotherapy research platform (SARRP, Xstrahl Inc.) focusing on the small field dosimetry. Physical dosimetry was assessed using ion chambers and radiochromic film, investigating the impact the beam focus size has on the dose rate output as well as beam characteristics (beam shape and penumbra variations). Two film reading modalities have been used to assess the dose output using the 0.5 mm diameter aperture. This study establishes that FilmQA Pro is a suitable tool for small field dosimetry, with a sufficiently small sampling area (0.1 mm) to ensure an accurate measurement. Furthermore, all small field dosimetry data generated in this study are equally comparable with our Monte Carlo simulations for both focal spot sizes. Overall, it was observed that the small focal spot, while having a lower dose-rate output was shown to produce a more stable and homogenous beam with minimum penumbra over time when compared to the larger focal spot. These findings suggest that when preclinical stereotactic irradiation fields are used, a practical compromise (time to deliver a desired dose and field homogeneity) needs to be considered when deciding the optimum treatment plan and beam configuration used. Citation Format: Jonathan L. Kane. Commissioning and radiobiological verification of the small animal radiotherapy research platform (SARRP) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4153.

Small field dosimetry for the small animal radiotherapy research platform (SARRP)

BackgroundPreclinical radiation biology has become increasingly sophisticated due to the implementation of advanced small animal image guided radiation platforms into laboratory investigation. These small animal radiotherapy devices enable state-of-the-art image guided therapy (IGRT) research to be performed by combining high-resolution cone beam computed tomography (CBCT) imaging with an isocentric irradiation system. Such platforms are capable of replicating modern clinical systems similar to those that integrate a linear accelerator with on-board CBCT image guidance.MethodsIn this study, we present a dosimetric evaluation of the small animal radiotherapy research platform (SARRP, Xstrahl Inc.) focusing on small field dosimetry. Physical dosimetry was assessed using ion chamber for calibration and radiochromic film, investigating the impact of beam focus size on the dose rate output as well as beam characteristics (beam shape and penumbra). Two film analysis tools) have been used to assess the dose output using the 0.5 mm diameter aperture.ResultsGood agreement (between 1.7–3%) was found between the measured physical doses and the data provided by Xstrahl for all apertures used. Furthermore, all small field dosimetry data are in good agreement for both film reading methods and with our Monte Carlo simulations for both focal spot sizes. Furthermore, the small focal spot has been shown to produce a more homogenous beam with more stable penumbra over time.ConclusionsFilmQA Pro is a suitable tool for small field dosimetry, with a sufficiently small sampling area (0.1 mm) to ensure an accurate measurement. The electron beam focus should be chosen with care as this can potentially impact on beam stability and reproducibility.

Abstract 835: Sensitivity of PTEN deficient non-small cell lung cancer to ionising radiation through inhibition of ataxia terangiectasia related 3 kinase (ATR)

Abstract This study sought to determine the therapeutic efficacy of AZD6738, a small molecule inhibitor of ataxia telangiectasia related 3 kinase (ATR), in combination with ionising radiation (IR) for the treatment of PTEN deficient non-small cell lung cancer (NSCLC). An isogenic PTEN deficient cell model were generated in H460 and A549 cell lines by HuSH PTEN shRNA constructs in pGFP-V-RS vectors from Origene. Radiosensitivity was determined at various concentrations of AZD6738 by clonogenic assay following IR with 225 kV X-rays. Target validation and mechanistic evaluation of the ATR radiation treatments was performed by western blotting. Cell cycle distribution was obtained using propidium iodine flow cytometry analysis which was performed 48 hours following treatment with AZD6738 alone or in combination with IR. In vivo efficacy was determined using cell line derived xenografts in female SCID mice treated with AZD6738 at a concentration of 25 mg/kg per day by oral gavage for 28 days. Animals were exposed to size fraction and hypofractionated radiation dose under CBCT image guidance using the small animal radiotherapy research platform (SARRP). Radiation induced toxicity in normal lung tissue effects was investigated in a model of radiation induced fibrosis in C57BL6/J mice. In-vitro, combined treatment with AZD6738 and IR selectively targets PTEN-deficient tumour cells causing a significant reduction in clonogenic survival for both H460- and A549-PTEN KO cell models (p= &amp;lt;0.05 at 8Gy). Following 2 Gy IR exposure there were significantly higher mean foci per cell in both H460-KO and A549-KO cells in comparison to WT cells (p= &amp;lt;0.05 at 2, 4 and 8 hours following treatment). Both PTEN-KO cell lines exhibited increased sub G0/G1 and G2/M populations in comparison to PTEN-WT cell lines when treated with AZD6738 and IR. Minimal toxicity was observed in both normal HBE and BEAS-2B cell lines. Currently in vivo tumour response experiments are underway to investigate IR and ATR kinase inhibitor AZD6738 using H460-WT and H460-PTEN KO cell models as well as determining the effects of AZD6738 in a radiation induced model of lung fibrosis. Approximately 10% of NSCLC patients have mutations in PTEN. This research therefore elucidates PTEN loss as a therapeutic target for combined IR with pharmacological inhibition of ATR in NSCLC. Targeting ATR inhibition in PTEN-deficient NSCLC may serve to increase radiation sensitivity in an in vivo model thus highlighting its clinical relevance as a potential personalised medicine approach for patients with PTEN deficient NSCLC. Citation Format: Victoria Louise Dunne, Kelly Redmond, Caroline Coffey, Karl Butterworth, Kevin Prise, Gerard Hanna. Sensitivity of PTEN deficient non-small cell lung cancer to ionising radiation through inhibition of ataxia terangiectasia related 3 kinase (ATR) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 835. doi:10.1158/1538-7445.AM2017-835

Modelling responses to spatially fractionated radiation fields using preclinical image-guided radiotherapy.

Radiotherapy is planned to achieve the optimal physical dose distribution to the target tumour volume whilst minimizing dose to the surrounding normal tissue. Recent in vitro experimental evidence has demonstrated an important role for intercellular communication in radiobiological responses following non-uniform exposures. This study aimed to model the impact of these effects in the context of techniques involving highly modulated radiation fields or spatially fractionated treatments such as spatially fractionated radiotherapy (GRID). Using the small-animal radiotherapy research platform as a key enabling technology to deliver precision imaged-guided radiotherapy, it is possible to achieve spatially modulated dose distributions that model typical clinical scenarios. In this work, we planned uniform and spatially fractionated dose distributions using multiple isocentres with beam sizes of 0.5-5 mm to obtain 50% volume coverage in a subcutaneous murine tumour model and applied a model of cellular response that incorporates intercellular communication to assess the potential impact of signalling effects with different ranges. Models of GRID treatment plans which incorporate intercellular signalling showed increased cell killing within the low-dose region. This results in an increase in the equivalent uniform dose for GRID exposures compared with standard models, with some GRID exposures being predicted to be more effective than uniform delivery of the same physical dose. This study demonstrates the potential impact of radiation-induced signalling on tumour cell response for spatially fractionated therapies and identifies key experiments to validate this model and quantify these effects in vivo. Advances in knowledge: This study highlights the unique opportunities now possible using advanced preclinical techniques to develop a foundation for biophysical optimization in radiotherapy treatment planning.

SU‐F‐T‐508: A Collimator‐Based 3‐Dimensional Grid Therapy Technique in a Small Animal Radiation Research Platform

Purpose:Three dimensional (3D) Grid Therapy using MLC‐based inverse‐planning has been proposed to achieve the features of both conformal radiotherapy and spatially fractionated radiotherapy, which may deliver very high dose in a single fraction to portions of a large tumor with relatively low normal tissue dose. However, the technique requires relatively long delivery time. This study aims to develop a collimator‐based 3D grid therapy technique. Here we report the development of the technique in a small animal radiation research platform.Methods:Similar as in the MLC‐based technique, 9 non‐coplanar beams in special channeling directions were used for the 3D grid therapy technique. Two specially designed grid collimators were fabricated, and one of them was selectively used to match the corresponding gantry/couch angles so that the grid opening of all 9 beams are met in the 3D space in the target. A stack of EBT3 films were used as 3D dosimetry to demonstrate the 3D grid‐like dose distribution in the target. Three 1‐mm beams were delivered to the stack of films in the area outside the target for alignment when all the films were scanned to reconstruct the 3D dosimtric image.Results:3D film dosimetry showed a lattice‐like dose distribution in the 3D target as well as in the axial, sagittal and coronal planes. The dose outside the target also showed a grid like dose distribution, and the average dose gradually decreased with the distance to the target. The peak to valley ratio was approximately 5:1. The delivery time was 7 minutes for 18 Gy peak dose, comparing to 6 minutes to deliver a 18‐Gy 3D conformal plan.Conclusion:We have demonstrated the feasibility of the collimator‐based 3D grid therapy technique which can significantly reduce delivery time comparing to MLC‐based inverse planning technique.

SU‐F‐T‐59: The Effect of Radiotherapy Dose On Immunoadjuvants

Purpose:Combining radiotherapy with immunotherapy is a promising approach to enhance treatment outcomes for cancer patients. This in‐vitro study investigated which radiotherapy doses could adversely affect the function of anti‐CD40 mAb, which is one of the key immunoadjuvants under investigations for priming such combination therapy.Methods:Human monocyte derived THP‐1 cells were treated with 100ng/mL of PMA in chamber slides to differentiate into macrophage. The THP‐1 differentiated macrophages were treated with 2uL/ml of the anti‐CD40 mAb and incubated at 37°C and 5% CO2 for 24 hours. Anti‐CD40 mAb treated cells were then irradiated at different doses of x‐rays: (0, 2, 4, 6, 8, and 12) Gy using the Small Animal Radiotherapy Research Platform (SARRP). After radiation, the cells were left at 4°C for 2 hours followed by immunofluorescence assay. A Nikon inverted live‐cell imaging system with fluorescence microscope was used to image the cells mounted on a slide fixed with Dapi. For comparison, an ELISA assay was performed with the antibody added to 3mL of PBS in multiple 10mm dishes. The 10mm dishes were irradiated at different x‐ray dose: (0, 2, 4, 6, 8. 10, 12, and 15) Gy using the SARRP.Results:The anti‐CD40 mAb activating the macrophages starts to lose their viability due to radiation dose between 8Gy to 12Gy as indicated by the immunofluorescence assay. The ELISA assay, also indicated that such high doses could lead to loss of the mAb's viability.Conclusion:This work suggests that high doses like those employed during Stereotactic Ablative Radiotherapy may affect the viability of immunoadjuvants such as anti‐CD 40. This study avails in‐vivo experiments combining radiotherapy with anti‐cd40 to get synergistic outcomes, including in the treatment of metastatic disease.