Small Animal Radiation Research Platform Research Articles

Response characterization of radiochromic OC-1 films in photon, electron, and proton beams.

Radiochromic film (RCF) dosimeters with their high spatial resolution and tissue equivalent properties are conveniently used for two-dimensional and small-field dosimetry. OC-1 is a new model of RCF dosimeter that was commercially introduced recently. Due to its novelty there is a need to characterize its response in various radiation beam types. To study the response of OC-1 RCFs to megavoltage clinical x-ray, electron, and proton beams, as well as kilovoltage x-ray beams used in a small animal research irradiator. OC-1 RCFs were cut into ∼4×4 cm2 pieces. RCF samples were irradiated at various dose levels in the range 0.5-120Gy using different modalities; a small animal radiation research platform (SARRP) (220 kVp), a medical linear accelerator (6 MV, 10 MV, 15 MV, 6 MV FFF, 10 MV FFF photon beams, as well as 6 and 20 MeV electron beams), and a gantry-mounted proton therapy synchrocyclotron. In order to study any dependency on the fractionation scheme, same dose was delivered at several fractions to a set of films. Different dose rates in the range 200-600 MU/min were delivered to a set of films to investigate any dose rate dependency. The films were scanned pre-irradiation and at 48h post-irradiation using a flatbed scanner. The net optical density (OD) was measured for red, green, and blue color channel for each film. The orientation dependency was studied by scanning the films at eight different orientations. In order to study the temporal evolution of the response of the films, film samples were irradiated at 10 and 50Gy using 6 MV photon beams and were scanned upon irradiation at certain time intervals up to 3 months. The spectral response of the films were studied over the visible range using a spectrometer. For megavoltage photon, electron, and plateau region of the proton beams, we did not observe a significant dependency on the beam quality, dose rate, and fractionation scheme. At the kV beam, an unusual over-response was observed in the films' net OD. An orientation dependency in the response of the films with a sinusoidal trend was observed. The response of the films increased with time following a double or triple exponential trend. The spectral absorption peaks were blue-shifted with dose. OC-1 RCFs were found to be reliable dosimeters with no significant energy dependency in MV range for photon and electron beams including the FFF beams. They over-respond when irradiated bykV x-ray beams compared to MV x-ray beams. Caution must be exercised to maintain the orientation of the films when scanning. Due to the temporal growth in the net OD of the films, same post-irradiation time interval must be used for scanning the calibration and test films.

Uncovering the connection between Ly6ChiCD103+ myeloid cells and radiation-induced cardiac fibrosis by CyTOF

Several immune cell populations participate in the development of radiation-induced cardiac fibrosis. However, the underlying mechanism remains to be elucidated. Male C57BL/6 mice (8–12 weeks old) were anesthetized and exposed to a single cardiac radiation dose of 20 Gy using the small-animal radiation research platform. We conducted echocardiography and histological analysis to identify cardiac fibrosis from a functional and pathological perspective. Time-of-flight mass cytometry (CyTOF) was used to describe the compositional changes of immune cells 1, 4, and 8 weeks after cardiac irradiation in mouse peripheral blood and cardiac tissue samples. Real-time quantitative polymerase chain reaction (RT-qPCR) was used to measure CD14 and CD105 mRNA levels from peripheral blood cells in eight patients who received thoracic radiotherapy 6 months ago. We observed reduced cardiac systolic function and increased collagen deposition in cardiac tissue after irradiation. Transforming growth factor-β, tumor necrosis factor-α, and interleukin-6 expression significantly elevated in the plasma. We identified a significant increase in CD45+ immune cell levels four weeks after cardiac irradiation using CyTOF, and neutrophil, monocyte, and macrophage levels elevated in blood samples after cardiac irradiation (four weeks). In cardiac tissue samples, monocyte and macrophage levels increased after cardiac irradiation (four weeks) compared with control group. Macrophages and monocytes were further analyzed, and the proportion of Ly6ChiCD103+ myeloid cells increased after cardiac irradiation in tissue samples (four weeks). RT-qPCR revealed that CD14 expression was higher in patients with cardiac injured group than in the control group (Previous studies reported that Ly6Chi monocytes in mice are similar to CD14 + CD16− monocytes in humans). We found that Ly6ChiCD103+ myeloid cells are critical subsets in radiation-induced cardiac fibrosis and might play protective roles in the process. Ly6ChiCD103+ myeloid cells may be considered an early biomarker for detecting radiation-induced cardiac fibrosis.

QOL-04.IN VIVO MODELLING RECAPITULATES RADIOTHERAPY DELIVERY AND LATE-EFFECT PROFILE FOR CHILDHOOD MEDULLOBLASTOMA

Abstract BACKGROUND Medulloblastoma (MB) is the most common malignant paediatric brain tumour, with 5-year survival rates >70%. Survivors frequently suffer a wide-range of late-effects due to their tumour and its treatment. Cranial radiotherapy (CRT) to the whole-brain, with posterior fossa boost (PFB), underpins treatment for non-infants, however, radiotherapeutic insult to normal brain tissue has deleterious consequences to neurocognitive and physical functioning, and causes accelerated ageing/frailty. Unfortunately, approaches to ameliorate radiotherapy-induced late-effects are lacking and a paucity of appropriate model systems hinders their development. METHODS We have developed a clinically-relevant in vivo model system that recapitulates the radiotherapy dose, delivery and late-effect profile of childhood medulloblastoma, at an equivalent developmental stage. Consistent with human regimens, age-equivalent (postnatal days 28-37) C57Bl/6J mice received targeted, CT image-guided, CRT (human-equivalent 38 Gy, n=12) or CRT with PFB (human-equivalent 49 Gy, n=12), via the small animal radiation research platform (SARRP) and were longitudinally assessed for over a year. Late-effects were compared to a sham-irradiated group (n=12). RESULTS CRT was well tolerated, independent of PFB receipt, and no mice suffered severe acute toxicity. Mice were significantly more frail following irradiation (frailty index; p=0.0002) and had reduced physical functioning; time to fall from a rotating rod and grip strength were significantly lower (rotarod; p=0.026 and grip strength; p=0.006, respectively). Neurocognitive deficits were consistent with those observed in childhood MB survivors; irradiated mice displayed significantly worse working memory (Y-maze; p=0.009) and exhibited spatial memory deficits (Barnes maze; p=0.029). Receipt of PFB did not induce a more severe late-effect profile. CONCLUSION We conclude that our in vivo model of childhood MB radiotherapy recapitulates the late-effect profile of MB survivors. Our clinically-relevant model will facilitate both the elucidation of novel/target mechanisms underpinning MB late-effects and the development of novel interventions for their amelioration.

Focused ultrasound-mediated blood–brain barrier opening is safe and feasible with moderately hypofractionated radiotherapy for brainstem diffuse midline glioma

BackgroundDiffuse midline glioma (DMG) is a pediatric tumor with dismal prognosis. Systemic strategies have been unsuccessful and radiotherapy (RT) remains the standard-of-care. A central impediment to treatment is the blood–brain barrier (BBB), which precludes drug delivery to the central nervous system (CNS). Focused ultrasound (FUS) with microbubbles can transiently and non-invasively disrupt the BBB to enhance drug delivery. This study aimed to determine the feasibility of brainstem FUS in combination with clinical doses of RT. We hypothesized that FUS-mediated BBB-opening (BBBO) is safe and feasible with 39 Gy RT.MethodsTo establish a safety timeline, we administered FUS to the brainstem of non-tumor bearing mice concurrent with or adjuvant to RT; our findings were validated in a syngeneic brainstem murine model of DMG receiving repeated sonication concurrent with RT. The brainstems of male B6 (Cg)-Tyrc-2J/J albino mice were intracranially injected with mouse DMG cells (PDGFB+, H3.3K27M, p53−/−). A clinical RT dose of 39 Gy in 13 fractions (39 Gy/13fx) was delivered using the Small Animal Radiation Research Platform (SARRP) or XRAD-320 irradiator. FUS was administered via a 0.5 MHz transducer, with BBBO and tumor volume monitored by magnetic resonance imaging (MRI).ResultsFUS-mediated BBBO did not affect cardiorespiratory rate, motor function, or tissue integrity in non-tumor bearing mice receiving RT. Tumor-bearing mice tolerated repeated brainstem BBBO concurrent with RT. 39 Gy/13fx offered local control, though disease progression occurred 3–4 weeks post-RT.ConclusionRepeated FUS-mediated BBBO is safe and feasible concurrent with RT. In our syngeneic DMG murine model, progression occurs, serving as an ideal model for future combination testing with RT and FUS-mediated drug delivery.

Abstract 699: Targeting radiation-driven invasion and metastasis in pancreatic cancer

Abstract Pancreatic ductal adenocarcinoma (PDAC) is an aggressive cancer with a dismal prognosis. Standard of care includes chemotherapy and radiotherapy (RT) for locally advanced, unresectable disease. Despite radiotherapy being an effective and sometimes curative option for many cancer types, radio-resistance is common, and pre-clinical research in recent years has found that RT can initiate a more aggressive, pro-invasive phenotype in the remaining cells post treatment (1,2). Little is known about the molecular mechanisms responsible for this phenomenon.There is accumulating evidence highlighting a role for the RhoGTPase family in mediating radioresistance and enhanced invasion (2, 3). Understanding their role in PDAC progression may aid the identification of novel chemotherapeutics that could be used alongside RT in the future. We present in vitro data that confirms the pro-migratory effect of RT and importantly indicates a significant increase in metastases formed in a clinically relevant model of PDAC (KPC Pdx1-Cre; KrasG12D/+; Trp53R172H/+) after treatment using a small animal radiation research platform (n=31, p<0.0001). In concurrence with previous literature, we found that PDAC cell motility was dependent on the RhoGTPase pathway, RhoA/ROCK1 (p=0.004). However, post-RT, this dependency was switched to an alternative pathway, CDC42/Rac1/MRCK, indicating a clear change in the balance of RhoGTPase signaling (p=0.002). This switch was confirmed via high-throughput immunofluorescence analysis. To widen this investigation further, we performed an siRNA screen targeting all members of the RhoGTPase family. Preliminary results indicate signaling dependency on Rac2, Rac3 and CDC42 post-RT. Additionally, we have performed RNAseq to investigate pro-invasive gene signatures arising from the acute irradiation of pancreatic cancer cells. This was done at sufficient depth to identify splice variants, with differential transcript expression (DTE) analysis identifying 236 differentially expressed transcripts and the differential transcript usage (DTU) analysis identifying 28 transcripts and 23 genes. Initial functional enrichment analysis found gene signatures relating to cell migration and adhesion, which may be indicative of an invasive phenotype. In conclusion, we present in vitro and in vivo data suggesting RT may promote a more invasive phenotype in PDAC at least in part due to a switch in RhoGTPase signaling. 1. Mueller, A.C., et al., Induction of ADAM10 by Radiation Therapy Drives Fibrosis, Resistance, and Epithelial-to-Mesenchyal Transition in Pancreatic Cancer. Cancer Res, 2021. 81(12): p. 3255-3269. 2. Birch, J.L., et al., A Novel Small-Molecule Inhibitor of MRCK Prevents Radiation-Driven Invasion in Glioblastoma. Cancer Res, 2018. 78(22): p. 6509-6522. Citation Format: Kathy Mclay, Mathias Tesson, Dion Gilmour, Jennifer P. Morton, Joanna L. Birch. Targeting radiation-driven invasion and metastasis in pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 699.

Radiosensitization of Allogenic Subcutaneous C6 Glioma Model with Focused Ultrasound-Induced Mild Hyperthermia.

The radiosensitization potential of focused ultrasound (FUS)-induced mild hyperthermia was assessed in an allogenic subcutaneous C6 glioma tumor model in rats. Mild hyperthermia at 42 °C was induced in tumors using a single-element 350 kHz FUS transducer. Radiation was delivered with a small animal radiation research platform using a single-beam irradiation technique. The combined treatment involved 20 min of FUS hyperthermia immediately before radiation. Tumor growth changes were observed one week post-treatment. A radiation dose of 2 Gy alone showed limited tumor control (30% reduction). However, when combined with FUS hyperthermia, there was a significant reduction in tumor growth compared to other treatments (tumor volumes: control-1174 ± 554 mm3, FUS-HT-1483 ± 702 mm3, 2 Gy-609 ± 300 mm3, FUS-HT + 2 Gy-259 ± 186 mm3; ANOVA p < 0.00001). Immunohistological analysis suggested increased DNA damage as a short-term mechanism for tumor control in the combined treatment. In conclusion, FUS-induced mild hyperthermia can enhance the effectiveness of radiation in a glioma tumor model, potentially improving the outcome of standard radiation treatments for better tumor control.

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.

In vivo modeling recapitulates radiotherapy delivery and late-effect profile for childhood medulloblastoma.

Medulloblastoma (MB) is the most common malignant pediatric brain tumor, with 5-year survival rates > 70%. Cranial radiotherapy (CRT) to the whole brain, with posterior fossa boost (PFB), underpins treatment for non-infants; however, radiotherapeutic insult to the normal brain has deleterious consequences to neurocognitive and physical functioning, and causes accelerated aging/frailty. Approaches to ameliorate radiotherapy-induced late-effects are lacking and a paucity of appropriate model systems hinders their development. We have developed a clinically relevant in vivo model system that recapitulates the radiotherapy dose, targeting, and developmental stage of childhood medulloblastoma. Consistent with human regimens, age-equivalent (postnatal days 35-37) male C57Bl/6J mice received computerized tomography image-guided CRT (human-equivalent 37.5 Gy EQD2, n = 12) ± PFB (human-equivalent 48.7 Gy EQD2, n = 12), via the small animal radiation research platform and were longitudinally assessed for > 12 months. CRT was well tolerated, independent of PFB receipt. Compared to a sham-irradiated group (n = 12), irradiated mice were significantly frailer following irradiation (frailty index; P = .0002) and had reduced physical functioning; time to fall from a rotating rod (rotarod; P = .026) and grip strength (P = .006) were significantly lower. Neurocognitive deficits were consistent with childhood MB survivors; irradiated mice displayed significantly worse working memory (Y-maze; P = .009) and exhibited spatial memory deficits (Barnes maze; P = .029). Receipt of PFB did not induce a more severe late-effect profile. Our in vivo model mirrored childhood MB radiotherapy and recapitulated features observed in the late-effect profile of MB survivors. Our clinically relevant model will facilitate both the elucidation of novel/target mechanisms underpinning MB late effects and the development of novel interventions for their amelioration.

Radiotherapy protocols for mouse cancer model.

Radiotherapy is a crucial treatment modality for cancer patients, with approximately 60% of individuals undergoing ionizing radiation as part of their disease management. In recent years, there has been a growing trend toward minimizing irradiation fields through the use of image-guided dosimetry and innovative technologies. These advancements allow for selective irradiation, delivering higher local doses while reducing the number of treatment sessions. Consequently, computer-assisted methods have significantly enhanced the effectiveness of radiotherapy in the curative and palliative treatment of various cancers. Although radiation therapy alone can effectively achieve local control in some cancer types, it may not be sufficient for others. As a result, further preclinical research is necessary to explore novel approaches including new schedules of radiotherapy treatments. Unfortunately, there is a concerning lack of correlation between clinical outcomes and experiments conducted on mouse models. We hypothesize that this disparity arises from the differences in irradiation strategies employed in preclinical studies compared to those used in clinical practice, which ultimately affects the translatability of findings to patients. In this study, we present two comprehensive radiotherapy protocols for the treatment of orthotopic melanoma and glioblastoma tumors. These protocols utilize a small animal radiation research platform, which is an ideal radiation device for delivering localized and precise X-ray doses to the tumor mass. By employing these platforms, we aim to limit the side effects associated with irradiating healthy surrounding tissues. Our detailed protocols offer a valuable framework for conducting preclinical studies that closely mimic clinical radiotherapy techniques, bridging the gap between experimental results and patient outcomes.

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.

TFAM Modulates Cardiomyocytes Pyroptosis Induced by Ionizing Radiation through mtDNA/TLR9/NF-kB Pathway

Mitochondrial transcription factor A (TFAM) is a pivotal factor for regulating mitochondrial DNA (mtDNA) replication, transcription and biogenesis. Previous studies have reported that cytosolic mtDNA stress can lead to cardiomyocytes pyroptosis, which is characterized by inflammasome formation. In this study, we attempted to investigate the mechanism of TFAM regulate cardiomyocytes pyroptosis induced by ionizing radiation. The peripheral blood serum of patients with esophageal cancer before and after definitive chemoradiotherapy was collected for Luminex multiplex cytokine assays. C57BL/6 mice were irradiated with the whole heart using small animal radiation research platform (SARRP) to construct a radiation-induced myocardial damage (RIMD) mouse model, and the ventricular function was evaluated using 9.4T Bruker magnetic resonance (MR) scanner. The function changes of cardiomyocytes exposed to radiation were observed in vitro and in vivo after knocking out GSDMD. Furthermore, the changes of mitochondrial function, the levels of cytosolic mtDNA, and the protein levels of NF-kB and pyroptosis pathway in irradiated cardiomyocytes were analyzed by knockdown and overexpression of TFAM in vitro and in vivo. By multifactor cytokine assays we found that pyroptosis related IL-1β and IL-18 were significantly increased in patients with high mean heart dose (MHD) after radiotherapy, while those with low MHD were not significantly increased after radiotherapy. Next, we successfully constructed the RIMD mouse model using a single heart irradiation of 20 Gy. We found that the gene expression of pyroptosis pathway was significantly up-regulated after cardiac irradiation by myocardial tissue transcriptomic sequencing. Compared with wild-type (WT) mice, cardiac systolic function of Gsdmd-/- mice was significantly improved at 1, 2, 6, 12, and 24 weeks after heart irradiation. In vitro, we also demonstrated increased viability of irradiated cardiomyocytes by knocking out GSDMD. In vitro and in vivo experiments confirmed the expression of TFAM decreased after radiation. By overexpression of TFAM, we found that irradiated cardiomyocytes showed improved mitochondrial function, decreased release of mtDNA into cytoplasm through mitochondrial permeability transition pores (mPTPs), decreased binding of cytosolic mtDNA to TLR9, and decreased expression of NF-kB and pyroptosis pathway proteins. Dual luciferase gene reporter assays and Chromatin immunoprecipitation (CHIP) assay confirmed that p65 could bind the NLRP3 promoter region. In addition, we found that ventricular function deteriorated and improved in mice with knockdown and overexpression of TFAM through adeno-associated virus serotype 9 (AAV9), respectively. Our study indicated that TFAM regulate irradiated cardiomyocytes pyroptosis through mtDNA/TLR9/NF-kB pathway. We provide a novel mechanism of RIMD, revealing an underappreciated intervention target for RIMD.

The Updated Landscape of Tumor Microenvironment and Pre-Metastasis Niches for Radiotherapy Resistant Tumors

In the current clinical work, hypo-fractionated radiotherapy (HFRT) and conventional fractionated radiotherapy (CFRT) are considered to be completely different treatments. However, the difference between the HFRT and CFRT in reprogramming the tumor immune microenvironment (TIME) is still inconclusive. Our previous work found that compared with HFRT, CFRT is more inclined to trigger immunosuppressive phenotype, but the underlying mechanism is still unclear. Based on this, we use single-cell mRNA sequencing to describe the landscape of TIME with HFRT and CFRT, and find the key targets or pathways to combine with radiotherapy. By simulating the in vivo radiotherapy strategy, we built recurrent murine cell lines mimicking CFRT and HFRT recurrent tumors (CFRT_R and HFRT_R tumors) with small animal radiation research platform (SARRP). Based on this unique model, we found that CFRT_R tumors can promote local relapse and lung metastasis significantly compared to HFRT_R tumors. Results of single-cell mRNA sequencing and FCAS also indicate that CFRT_R tumors possess more macrophages in the tumor site and neutrophils in the metastatic site. By using quantitative proteomics of secreted proteins, we prove that CFRT_R tumors secret more Ccl2, S100a11 and Slpi. Mechanically, CFRT_R tumors influence the polarization of M2 TAMs leading to tumor growth through secretion of Ccl2 and S100a11. Meanwhile, CFRT_R tumors also augment the infiltration of neutrophils in the lung through Slpi altering the lung immune landscape to support metastasis. ChIP and EMSA results also reveal that RelB is the core transcription factor of Ccl2, S100a11 and Slpi. Furthermore, local relapse and lung metastasis can be reversed by targeting non-canonical NF-kB pathway. The effect of CFRT_R and HFRT_R tumors in reprogramming TIME is different, CFRT_R tumors can trigger immunosuppressive phenotype compared to HFRT_R tumors by possessing more M2 TAMs in the tumor site and neutrophils in the metastatic site. The killing function of tumor infiltrated T cells can be inhibited by accumulated M2 TAMs, and abundant neutrophils in lungs contribute to the formation of the pre-metastatic niche. Targeting non-canonical NF-kB pathway can reverse local relapse and lung metastasis in CFRT_R tumors by suppressing Ccl2, S100a11 and Slpi secretion. In short, our study not only deepens the understanding of the immune landscape of CFRT and HFRT recurrence tumors, but also provides a novel therapy of CFRT recurrence patients.

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

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. 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. 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. 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 13-18mm above the mouse bed. The spatial resolution of 2D optical imaging is<0.3mm at Rayleigh criterion. Within the region of interest, the image uniformity is within 5% variation, and image shift due to distortion is within 0.3mm. 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 0.7mm. Our phantom test shows the BLT system can localize a bioluminescent target within 1mm with an optimal threshold and only 0.2mm 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. 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.

Triple‑negative breast cancer cells that survive ionizing radiation exhibit an Axl‑dependent aggressive radioresistant phenotype.

This study aimed to investigate the aggressive behavior of triple-negative breast cancer (TNBC) cells that had survived ionizing radiation and explore the potential targets of TNBC combination treatment. Consistent with the previous literature, Axl was highly expressed in TNBC and closely associated with the degree of malignancy based on immunohistochemical staining. Using a gradient irradiation method, the ionizing radiation-resistant mouse TNBC cell line 4T-1/IRR was established. It was found that Axl expression was upregulated in 4T-1/IRR cells. After irradiation by X-ray, the cell viability and colony formation ability of 4T-1/IRR cells were significantly increased when compared with the 4T-1 cells. Combined radiotherapy with Axl inhibition by treatment with R428 and small interfering RNA lentivirus targeting Axl infection significantly reduced cell viability, colony formation ability, DNA double-stranded break repair, and the invasive and migratory ability of 4T-1/IRR cells. In vivo, the small animal radiation research platform was applied to precisely administer radiotherapy of the tumor-bearing mice. R428 treatment combined with 6 Gy X-ray significantly inhibited the growth of 4T-1/IRR cells-derived xenograft tumors in the BALB/c mouse. The results of western blotting showed that the critical molecular mechanism involved in the radioresistance of TNBC cells was the PI3K/Akt/mTOR signaling pathway induced by Axl activation. Thus, it is hypothesized that targeted Axl therapy combined with radiotherapy may have significant potential for the treatment of TNBC.

Early Normal Tissue Effects and Bone Marrow Relative Biological Effectiveness for an Actinium 225-Labeled HER2/neu-Targeting Antibody

In this study we have determined the dose-independent relative biological effectiveness (RBE2) of bone marrow for an anti-HER2/neu antibody labeled with the alpha-particle emitter actinium-225 (225Ac). Hematologic toxicity is often a consequence of radiopharmaceutical therapy (RPT) administration, and dosimetric guidance to the bone marrow is required to limit toxicity. Female neu/N transgenic mice (MMTV-neu) were intravenously injected with 0-16.65 kBq of the alpha-particle emitter labeled antibody, 225Ac-DOTA-7.16.4 and sacrificed at 1-9 days after-treatment. Complete blood counts (CBC) were performed. Femurs and tibias were collected, and bone marrow was isolated from one femur and tibia, and counted for radioactivity. Contralateral intact femurs were fixed, decalcified and assessed by histology. Marrow cellularity was the biological endpoint selected for RBE2 determination. For the reference radiation, both femurs of the mice were photon irradiated with 0-5 Gy, using a small animal radiation research platform. Response as measured by cellularity for the alpha-particle emitter RPT and the external beam radiotherapy were linear and linear quadratic, respectively, as a function of absorbed dose. The resulting dose-independent RBE2 for bone marrow was 6. As alpha-emitter RPT gains prominence, preclinical studies evaluating RBE, in vivo, will be important in relating to human experience with beta-particle emitter RPT. Such normal tissue RBE evaluations will help mitigate unexpected toxicity in αRPT.

Combining immunotherapy with high-dose radiation therapy (HDRT) significantly inhibits tumor growth in a syngeneic mouse model of high-risk neuroblastoma

PurposeThe mortality in patients with MYCN-amplified high-risk neuroblastoma remains greater than 50% despite advances in multimodal therapy. Novel therapies are urgently needed that requires preclinical evaluation in appropriate mice models. Combinatorial treatment with high-dose radiotherapy (HDRT) and immunotherapy has emerged as an effective treatment option in a variety of cancers. Current models of neuroblastoma do not recapitulate the anatomic and immune environment in which multimodal therapies can be effectively tested, and there is a need for an appropriate syngeneic neuroblastoma mice model to study interaction of immunotherapy with host immune cells. Here, we develop a novel syngeneic mouse model of MYCN-amplified neuroblastoma and report the relevance and opportunities of this model to study radiotherapy and immunotherapy. Materials and methodsA syngeneic allograft tumor model was developed using the murine neuroblastoma cell line 9464D derived a tumor from TH-MYCN transgenic mouse. Tumors were generated by transplanting 1 mm3 portions of 9464D flank tumors into the left kidney of C57Bl/6 mice. We investigated the effect of combining HDRT with anti-PD1 antibody on tumor growth and tumor microenvironment. HDRT (8 Gy x 3) was delivered by the small animal radiation research platform (SARRP). Tumor growth was monitored by ultrasound. To assess the effect on immune cells tumors sections were co-imuunostained for six biomarkers using the Vectra multispectral imaging platform. ResultsTumor growth was uniform and confined to the kidney in 100% of transplanted tumors. HDRT was largely restricted to the tumor region with minimal scattered out-of-field dose. Combinatorial treatment with HDRT and PD-1 blockade significantly inhibited tumor growth and prolonged mice survival. We observed augmented T-lymphocyte infiltration, especially CD3+CD8+ lymphocytes, in tumors of mice which received combination treatment. ConclusionWe have developed a novel syngeneic mouse model of MYCN amplified high-risk neuroblastoma. We have utilized this model to show that combining immunotherapy with HDRT inhibits tumor growth and prolongs mice survival.

Development of a PTV margin for preclinical irradiation of orthotopic pancreatic tumors derived from a well-known recipe for humans

In human radiotherapy a safety margin (PTV margin) is essential for successful irradiation and is usually part of clinical treatment planning. In preclinical radiotherapy research with small animals, most uncertainties and inaccuracies are present as well, but according to the literature a margin is used only scarcely. In addition, there is only little experience about the appropriate size of the margin, which should carefully be investigated and considered, since sparing of organs at risk or normal tissue is affected.Here we estimate the needed margin for preclinical irradiation by adapting a well-known human margin recipe from van Herck et al. to the dimensions and requirements of the specimen on a small animal radiation research platform (SARRP). We adjusted the factors of the described formula to the specific challenges in an orthotopic pancreatic tumor mouse model to establish an appropriate margin concept. The SARRP was used with its image-guidance irradiation possibility for arc irradiation with a field size of 10 × 10 mm2 for 5 fractions. Our goal was to irradiate the clinical target volume (CTV) of at least 90% of our mice with at least 95% of the prescribed dose. By carefully analyzing all relevant factors we gain a CTV to planning target volume (PTV) margin of 1.5 mm for our preclinical setup.The stated safety margin is strongly dependent on the exact setting of the experiment and has to be adjusted for other experimental settings. The few stated values in literature correspond well to our result. Even if using margins in the preclinical setting might be an additional challenge, we think it is crucial to use them to produce reliable results and improve the efficacy of radiotherapy.

Abstract 2839: Methionine restriction radiosensitizes KRAS mutant rectal cancer

Abstract Purpose/Objective(s): Radiation therapy (RT) is the standard of care in patients with locally advanced rectal cancer, but not all patients respond equally to RT. KRAS-mutated (KRASmut) cancer cells are highly radioresistance and require higher levels of methionine, an essential amino acid, compared to KRAS wildtype (KRASwt) and normal cells, and diet restriction of methionine has been shown to potentiate the effects of radiation in vivo. We hypothesized that methionine restriction (MR) could radiosensitize KRASmut rectal cancer cells. Materials/Methods: HCT116 KRASG13D/+ and SW48 KRASwt colorectal carcinoma isogenic cell lines and their isogenic derivative cell lines HCT 116 KRAS+/- and SW48 KRASG13D were obtained from Horizon Discovery Ltd (Cambridge, UK). Radiation clonogenic assays, immunofluorescence, and western blot analysis of key DNA damage response proteins were performed in the KRASmut vs. KRASwt colorectal cells treated with ionizing radiation (IR, 2-6 Gy) at increasing time points following pre-treatment with methionine control (MC) (200 µM) or MR (40-fold reduction in methionine, 5 µM) media for 48 hours. One hundred thousand luciferase-expressing KRASwt or KRASmut SW48 cells were implanted orthotopically in the rectum of athymic nude mice. After confirmation of tumor formation at one-week post-injection, the mouse diet was changed to either an MC (0.84% methionine w/w) or MR (0.12% methionine w/w) diet for a total of two weeks. Rectal tumors were treated on an Xstrahl small animal radiation research platform (SARRP) to a dose of 25 Gy in 5 daily fractions (1 cm x 1 cm collimator, isocenter at anal verge) during the second week of diet modification. Tumor growth was measured via bioluminescence on IVIS Lumina every two weeks for four weeks post-radiation. Results: MR significantly increased radiosensitization compared to MC in both HCT116 and SW48 cells in vitro. KRASmut cells showed a 20-30% increase in radiation in the dose enhancement ratio compared to the KRASwt cells. The protein expression and nuclear foci per cell of the DNA damage marker γH2AX were significantly higher in the KRASmut cells compared to KRASwt cells following 5 Gy of RT at 6 hours. Protein expression of homologous recombination pathway proteins RAD51 and the CtBP interacting protein (CtIP) were higher at baseline in the KRASmut cells than KRASwt. MR substantially reduced the expression of these proteins following RT. Mice harboring SW48 KRASmut rectal tumors treated with 5 Gy x 5 RT and MR showed a significant increase in the complete response rate compared to MC (80% vs. 10%, p=0.02). In the mice harboring SW48 KRASwt rectal tumors, there was no significant difference in response rate between MR vs. MC (40% vs. 30%, p=NS). Conclusion: Our study indicates a novel strategy to radiosensitize resistant KRAS-mutated rectal cancer through MR. Studies testing the mechanism of KRAS-dependent methionine-mediated DNA repair is ongoing. Citation Format: Adam R. Wolfe, Sarita Garg, Stetson Van Matre, Robin Eluvathingal, Oscar Zuniga, Henrique Rodrigues, Nükhet Aykin-Burns, Isabelle Racine Miousse. Methionine restriction radiosensitizes KRAS mutant rectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2839.

Abstract 1101: Combination therapy to overcome radiotherapy induced immunosuppression and improve tumor control

Abstract Background: The relationship between radiation therapy, immunosuppressive myeloid populations and adenosine is not well characterized. Here we test the hypothesis that radiation-induced adenosine signaling promotes tumor growth and causes suppression of T cell function and that therapeutic targeting may improve response to radiotherapy. Methods: Orthotopic 4T1 cells were implanted in Balb/c mice and tumors were irradiated using small animal radiation research platform (SARRP, Xstrahl; 220 kV) with 3 fractions of 8 Gy and/or treated with adenosine receptor 2A and 2B (A2AR/A2BRi) inhibitor, CD73 inhibitor (CD73i), and/or checkpoint inhibitor, aPD-1. Tumor response was assessed by tumor volume and the immune response characterized by flow cytometry. Results: Intratumoral immune response was evaluated 72 hours post-irradiation. Radiotherapy induced an expansion of myeloid cells particularly monocytic cells (13.5% vs 7% of CD45+ cells) with a corresponding contraction of lymphocytes. Monocytes from irradiated tumors had increased expression of most of the key members of the adenosine signaling pathway including enpp1 (2.5% vs 0.6%), CD73 (10.3% vs 4.8%), CD39 (10.7% vs 4.4%), A2aR (2.8% vs 0.6%) and A2bR (2.4% vs 0.3%), compared to untreated controls. In the absence of tumor irradiation, the combination of A2AR/A2BRi &amp; CD73i with αPD-1 showed no improvement in tumor control compared to A2AR/A2BRi &amp; CD73i alone or untreated controls. However, in the setting of tumor irradiation, we measured a significant reduction in 4T1 tumor volume when aPD-1 was added to A2AR/A2BRi &amp; CD73i, compared to A2AR/A2BRi &amp; CD73i alone (156mm^3 vs 250mm^3). The addition of αPD-1 to A2AR/A2BRi &amp; CD73i and tumor irradiation resulted in decrease in monocytic cells (9.4% vs 15% of CD45+ cells) and expression of CD39 (7.4% vs 11%), CD73 (7.5% vs 11%), A2aR (2.2% vs 7%) and A2bR (2.7% vs 9%) in monocytic compartment, compared to radiation and A2AR/A2BRi &amp; CD73i. Further, there was a corresponding expansion of Ki67+ proliferating (52% vs 36%) and IFN-γ producing CD8+ T cells (3.3% vs 2%) after treatment with radiation, A2AR/A2BRi &amp; CD73i, and αPD-1, suggesting improved T cell effector function. Conclusion: Radiation-induced adenosine signaling facilitates immunosuppressive changes in the TME. In the immunotherapy insensitive 4T1 mouse model, adenosine blockade induces checkpoint inhibition sensitivity through alleviation of immunosuppression, thus improving tumor control. Citation Format: Shruti Bansal, Matthew Chaimowitz, Julia Ann, Patrick McCann, Jason Piersaint, Claudia Aiello, Namita Sen, Julia Kogan, Catherine Spina. Combination therapy to overcome radiotherapy induced immunosuppression and improve tumor control [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1101.

Characterization of the Response of 9L and U-251N Orthotopic Brain Tumors to 3D Conformal Radiation Therapy.

In a study employing MRI-guided stereotactic radiotherapy (SRS) in two orthotopic rodent brain tumor models, the radiation dose yielding 50% survival (the TCD50) was sought. Syngeneic 9L cells, or human U-251N cells, were implanted stereotactically in 136 Fischer 344 rats or 98 RNU athymic rats, respectively. At approximately 7 days after implantation for 9L, and 18 days for U-251N, rats were imaged with contrast-enhanced MRI (CE-MRI) and then irradiated using a Small Animal Radiation Research Platform (SARRP) operating at 220 kV and 13 mA with an effective energy of ∼70 keV and dose rate of ∼2.5 Gy per min. Radiation doses were delivered as single fractions. Cone-beam CT images were acquired before irradiation, and tumor volumes were defined using co-registered CE-MRI images. Treatment planning using MuriPlan software defined four non-coplanar arcs with an identical isocenter, subsequently accomplished by the SARRP. Thus, the treatment workflow emulated that of current clinical practice. The study endpoint was animal survival to 200 days. The TCD50 inferred from Kaplan-Meier survival estimation was approximately 25 Gy for 9L tumors and below 20 Gy, but within the 95% confidence interval in U-251N tumors. Cox proportional-hazards modeling did not suggest an effect of sex, with the caveat of wide confidence intervals. Having identified the radiation dose at which approximately half of a group of animals was cured, the biological parameters that accompany radiation response can be examined.