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Purpose: Tumor hypoxia contributes to aggressive phenotypes and diminished therapeutic responses to radiation therapy (RT) with hypoxic tissue being 3-fold less radiosensitive than normoxic tissue. A major challenge in implementing hypoxic radiosensitizers is the lack of a high-resolution imaging modality that directly quantifies tissue-oxygen. The electron paramagnetic resonance oxygen-imager (EPROI) was used to quantify tumor oxygenation in two murine tumor models: E0771 syngeneic transplant breast cancers and primary p53/MCA soft tissue sarcomas, with the latter autochthonous model better recapitulating the tumor microenvironment in human malignancies. We hypothesized that tumor hypoxia differs between these models. We also aimed to quantify the absolute change in tumor hypoxia induced by the mitochondrial inhibitor papaverine (PPV) and its effect on RT response., Procedures: Tumor oxygenation was characterized in E0771 and primary p53/MCA sarcomas via EPROI, with the former model also being quantified indirectly via diffuse reflectance spectroscopy (DRS). After confirming PPV's effect on hypoxic fraction (via EPROI), we compared the effect of 0 versus 2 mg/kg PPV prior to 20 Gy on tumor growth delay and survival., Results: Hypoxic sarcomas were more radioresistant than normoxic sarcomas (p=0.0057, 2-way ANOVA), and high baseline hypoxic fraction was a significant (p=0.0063, Cox Regression Model) hazard in survivability regardless of treatment. Pre-treatment with PPV before RT did not radiosensitize tumors in the sarcoma or E0771 model. In the sarcoma model, EPROI successfully identified baseline hypoxic tumors. DRS quantification of total hemoglobin, saturated hemoglobin, changes in mitochondrial potential and glucose uptake showed no significant difference in E0771 tumors pre- and post-PPV., Conclusion: EPROI provides 3D high-resolution pO 2 quantification; EPR is better suited than DRS to characterize tumor hypoxia. PPV did not radiosensitize E0771 tumors nor p53/MCA sarcomas, which may be related to the complex pattern of vasculature in each tumor. Additionally, understanding model-dependent tumor hypoxia will provide a much-needed foundation for future therapeutic studies with hypoxic radiosensitizers., (© 2023. The Author(s), under exclusive licence to World Molecular Imaging Society.)

Purpose: To identify significant relationships between quantitative cytometric tissue features and quantitative MR (qMRI) intratumorally in preclinical undifferentiated pleomorphic sarcomas (UPS)., Materials and Methods: In a prospective study of genetically engineered mouse models of UPS, we registered imaging libraries consisting of matched multi-contrast in vivo MRI, three-dimensional (3D) multi-contrast high-resolution ex vivo MR histology (MRH), and two-dimensional (2D) tissue slides. From digitized histology we generated quantitative cytometric feature maps from whole-slide automated nuclear segmentation. We automatically segmented intratumoral regions of distinct qMRI values and measured corresponding cytometric features. Linear regression analysis was performed to compare intratumoral qMRI and tissue cytometric features, and results were corrected for multiple comparisons. Linear correlations between qMRI and cytometric features with p values of <0.05 after correction for multiple comparisons were considered significant., Results: Three features correlated with ex vivo apparent diffusion coefficient (ADC), and no features correlated with in vivo ADC. Six features demonstrated significant linear relationships with ex vivo T2*, and fifteen features correlated significantly with in vivo T2*. In both cases, nuclear Haralick texture features were the most prevalent type of feature correlated with T2*. A small group of nuclear topology features also correlated with one or both T2* contrasts, and positive trends were seen between T2* and nuclear size metrics., Conclusion: Registered multi-parametric imaging datasets can identify quantitative tissue features which contribute to UPS MR signal. T2* may provide quantitative information about nuclear morphology and pleomorphism, adding histological insights to radiological interpretation of UPS., Competing Interests: DK is a co-founder of XRAD Therapeutics, which is commercializing radiosensitizers. DK is on the scientific advisory board for Lumicell, which is commercializing intraoperative imaging technology. DK has received research support from Merck, XRAD Therapeutics, Bristol Myers Squibb, and Varian. However, this funding did not support this work. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Blocker, Mowery, Everitt, Cook, Cofer, Qi, Bassil, Xu, Kirsch, Badea and Johnson.)

Purpose: Despite aggressive multimodal treatment that typically includes definitive or adjuvant radiation therapy (RT), locoregional recurrence rates approach 50% for patients with locally advanced human papillomavirus (HPV)-negative head and neck squamous cell carcinoma (HNSCC). Thus, more effective therapeutics are needed to improve patient outcomes. We evaluated the radiosensitizing effects of ataxia telangiectasia and RAD3-related (ATR) inhibitor (ATRi) BAY 1895344 in preclinical models of HNSCC., Methods and Materials: Murine and human HPV-negative HNSCC cells (MOC2, MOC1, JHU-012) were treated with vehicle or ATRi with or without 4 Gy. Checkpoint kinase 1 phosphorylation and DNA damage (γH2AX) were evaluated by Western blot, and ATRi half-maximal inhibitory concentration was determined by MTT assay for HNSCC cells and immortalized murine oral keratinocytes. In vitro radiosensitization was tested by clonogenic assay. Cell cycle distribution and mitotic catastrophe were evaluated by flow cytometry. Mitotic aberrations were quantified by fluorescent microscopy. Tumor growth delay and survival were assessed in mice bearing MOC2 or JHU-012 transplant tumors treated with vehicle, ATRi, RT (10 Gy × 1 or 8 Gy × 3), or combined ATRi + RT., Results: ATRi caused dose-dependent reduction in checkpoint kinase 1 phosphorylation at 1 hour post-RT (4 Gy) and dose-dependent increase in γH2AX at 18 hours post-RT. Addition of RT to ATRi led to decreased BAY 1895344 half-maximal inhibitory concentration in HNSCC cell lines but not in normal tissue surrogate immortalized murine oral keratinocytes. Clonogenic assays demonstrated radiosensitization in the HNSCC cell lines. ATRi abrogated the RT-induced G2/M checkpoint, leading to mitosis with unrepaired DNA damage and increased mitotic aberrations (multinucleated cells, micronuclei, nuclear buds, nucleoplasmic bridges). ATRi and RT significantly delayed tumor growth in MOC2 and JHU-012 in vivo models, with improved overall survival in the MOC2 model., Conclusions: These findings demonstrated that BAY 1895344 increased in vitro and in vivo radiosensitivity in HPV-negative HNSCC preclinical models, suggesting therapeutic potential warranting evaluation in clinical trials for patients with locally advanced or recurrent HPV-negative HNSCC., (Copyright © 2023 Elsevier Inc. All rights reserved.)

This study aims to investigate whether adding neoadjuvant radiotherapy (RT), anti-programmed cell death protein-1 (PD-1) antibody (anti-PD-1), or RT + anti-PD-1 to surgical resection improves disease-free survival for mice with soft tissue sarcomas (STS). We generated a high mutational load primary mouse model of STS by intramuscular injection of adenovirus expressing Cas9 and guide RNA targeting Trp53 and intramuscular injection of 3-methylcholanthrene (MCA) into the gastrocnemius muscle of wild-type mice (p53/MCA model). We randomized tumor-bearing mice to receive isotype control or anti-PD-1 antibody with or without radiotherapy (20 Gy), followed by hind limb amputation. We used micro-CT to detect lung metastases with high spatial resolution, which was confirmed by histology. We investigated whether sarcoma metastasis was regulated by immunosurveillance by lymphocytes or tumor cell-intrinsic mechanisms. Compared with surgery with isotype control antibody, the combination of anti-PD-1, radiotherapy, and surgery improved local recurrence-free survival (P = 0.035) and disease-free survival (P = 0.005), but not metastasis-free survival. Mice treated with radiotherapy, but not anti-PD-1, showed significantly improved local recurrence-free survival and metastasis-free survival over surgery alone (P = 0.043 and P = 0.007, respectively). The overall metastasis rate was low (∼12%) in the p53/MCA sarcoma model, which limited the power to detect further improvement in metastasis-free survival with addition of anti-PD-1 therapy. Tail vein injections of sarcoma cells into immunocompetent mice suggested that impaired metastasis was due to inability of sarcoma cells to grow in the lungs rather than a consequence of immunosurveillance. In conclusion, neoadjuvant radiotherapy improves metastasis-free survival after surgery in a primary model of STS., (©2022 American Association for Cancer Research.)

The purpose of this study was to investigate if radiomic analysis based on spectral micro-CT with nanoparticle contrast-enhancement can differentiate tumors based on lymphocyte burden. High mutational load transplant soft tissue sarcomas were initiated in Rag2 +/- and Rag2 -/- mice to model varying lymphocyte burden. Mice received radiation therapy (20 Gy) to the tumor-bearing hind limb and were injected with a liposomal iodinated contrast agent. Five days later, animals underwent conventional micro-CT imaging using an energy integrating detector (EID) and spectral micro-CT imaging using a photon-counting detector (PCD). Tumor volumes and iodine uptakes were measured. The radiomic features (RF) were grouped into feature-spaces corresponding to EID, PCD, and spectral decomposition images. The RFs were ranked to reduce redundancy and increase relevance based on TL burden. A stratified repeated cross validation strategy was used to assess separation using a logistic regression classifier. Tumor iodine concentration was the only significantly different conventional tumor metric between Rag2 +/- (TLs present) and Rag2 -/- (TL-deficient) tumors. The RFs further enabled differentiation between Rag2 +/- and Rag2 -/- tumors. The PCD-derived RFs provided the highest accuracy (0.68) followed by decomposition-derived RFs (0.60) and the EID-derived RFs (0.58). Such non-invasive approaches could aid in tumor stratification for cancer therapy studies.

Objective . To develop and characterize novel methods of extreme spatially fractionated kV radiation therapy (including mini-GRID therapy) and to evaluate efficacy in the context of a pre-clinical mouse study. Approach . Spatially fractionated GRIDs were precision-milled from 3 mm thick lead sheets compatible with mounting on a 225 kVp small animal irradiator (X-Rad). Three pencil-beam GRIDs created arrays of 1 mm diameter beams, and three 'bar' GRIDs created 1 × 20 mm rectangular fields. GRIDs projected 20 × 20 mm 2 fields at isocenter, and beamlets were spaced at 1, 1.25, and 1.5 mm, respectively. Peak-to-valley ratios and dose distributions were evaluated with Gafchromic film. Syngeneic transplant tumors were induced by intramuscular injection of a soft tissue sarcoma cell line into the gastrocnemius muscle of C57BL/6 mice. Tumor-bearing mice were randomized to four groups: unirradiated control, conventional irradiation of entire tumor, GRID therapy, and hemi-irradiation (half-beam block, 50% tumor volume treated). All irradiated mice received a single fraction of 15 Gy. Results . High peak-to-valley ratios were achieved (bar GRIDs: 11.9 ± 0.9, 13.6 ± 0.4, 13.8 ± 0.5; pencil-beam GRIDs: 18.7 ± 0.6, 26.3 ± 1.5, 31.0 ± 3.3). Pencil-beam GRIDs could theoretically spare more intra-tumor immune cells than bar GRIDs, but they treat less tumor tissue (3%-4% versus 19%-23% area receiving 90% prescription, respectively). Bar GRID and hemi-irradiation treatments significantly delayed tumor growth ( P  < 0.05), but not as much as a conventional treatment ( P  < 0.001). No significant difference was found in tumor growth delay between GRID and hemi-irradiation. Significance . High peak-to-valley ratios were achieved with kV grids: two-to-five times higher than values reported in literature for MV grids. GRID irradiation and hemi-irradiation delayed tumor growth, but neither was as effective as conventional whole tumor uniform dose treatment. Single fraction GRID therapy could not initiate an anti-cancer immune response strong enough to match conventional RT outcomes, but follow-up studies will evaluate the combination of mini-GRID with immune checkpoint blockade., (© 2022 Institute of Physics and Engineering in Medicine.)

We are developing imaging methods for a co-clinical trial investigating synergy between immunotherapy and radiotherapy. We perform longitudinal micro-computed tomography (micro-CT) of mice to detect lung metastasis after treatment. This work explores deep learning (DL) as a fast approach for automated lung nodule detection. We used data from control mice both with and without primary lung tumors. To augment the number of training sets, we have simulated data using real augmented tumors inserted into micro-CT scans. We employed a convolutional neural network (CNN), trained with four competing types of training data: (1) simulated only, (2) real only, (3) simulated and real, and (4) pretraining on simulated followed with real data. We evaluated our model performance using precision and recall curves, as well as receiver operating curves (ROC) and their area under the curve (AUC). The AUC appears to be almost identical (0.76-0.77) for all four cases. However, the combination of real and synthetic data was shown to improve precision by 8%. Smaller tumors have lower rates of detection than larger ones, with networks trained on real data showing better performance. Our work suggests that DL is a promising approach for fast and relatively accurate detection of lung tumors in mice.

Purpose To establish a platform for quantitative tissue-based interpretation of cytoarchitecture features from tumor MRI measurements. Materials and Methods In a pilot preclinical study, multicontrast in vivo MRI of murine soft-tissue sarcomas in 10 mice, followed by ex vivo MRI of fixed tissues (termed MR histology ), was performed. Paraffin-embedded limb cross-sections were stained with hematoxylin-eosin, digitized, and registered with MRI. Registration was assessed by using binarized tumor maps and Dice similarity coefficients (DSCs). Quantitative cytometric feature maps from histologic slides were derived by using nuclear segmentation and compared with registered MRI, including apparent diffusion coefficients and transverse relaxation times as affected by magnetic field heterogeneity (T2* maps). Cytometric features were compared with each MR image individually by using simple linear regression analysis to identify the features of interest, and the goodness of fit was assessed on the basis of R 2 values. Results Registration of MR images to histopathologic slide images resulted in mean DSCs of 0.912 for ex vivo MR histology and 0.881 for in vivo MRI. Triplicate repeats showed high registration repeatability (mean DSC, >0.9). Whole-slide nuclear segmentations were automated to detect nuclei on histopathologic slides (DSC = 0.8), and feature maps were generated for correlative analysis with MR images. Notable trends were observed between cell density and in vivo apparent diffusion coefficients (best line fit: R 2 = 0.96, P < .001). Multiple cytoarchitectural features exhibited linear relationships with in vivo T2* maps, including nuclear circularity (best line fit: R 2 = 0.99, P < .001) and variance in nuclear circularity (best line fit: R 2 = 0.98, P < .001). Conclusion An infrastructure for registering and quantitatively comparing in vivo tumor MRI with traditional histologic analysis was successfully implemented in a preclinical pilot study of soft-tissue sarcomas. Keywords: MRI, Pathology, Animal Studies, Tissue Characterization Supplemental material is available for this article. © RSNA, 2021.

Immunotherapy fails to cure most cancer patients. Preclinical studies indicate that radiotherapy synergizes with immunotherapy, promoting radiation-induced antitumor immunity. Most preclinical immunotherapy studies utilize transplant tumor models, which overestimate patient responses. Here, we show that transplant sarcomas are cured by PD-1 blockade and radiotherapy, but identical treatment fails in autochthonous sarcomas, which demonstrate immunoediting, decreased neoantigen expression, and tumor-specific immune tolerance. We characterize tumor-infiltrating immune cells from transplant and primary tumors, revealing striking differences in their immune landscapes. Although radiotherapy remodels myeloid cells in both models, only transplant tumors are enriched for activated CD8+ T cells. The immune microenvironment of primary murine sarcomas resembles most human sarcomas, while transplant sarcomas resemble the most inflamed human sarcomas. These results identify distinct microenvironments in murine sarcomas that coevolve with the immune system and suggest that patients with a sarcoma immune phenotype similar to transplant tumors may benefit most from PD-1 blockade and radiotherapy.

Background: Tetracycline antibiotics inhibit matrix metalloproteinases and pro-inflammatory cytokines implicated in the pathogenesis of tendinopathy, while microsphere formulations allow sustained release of drug contents. The purpose of this study was to evaluate the ability of a local minocycline microsphere injection to restore normal tendon properties in a rat model of collagenase-induced patellar tendinopathy., Methods: A total of 22 rats were randomly assigned to the control (n = 11) or minocycline (n = 11) group and received bilateral patellar tendon injections of collagenase. After 7 days, the minocycline group received the minocycline microsphere treatment and the control group received phosphate buffered solution. Pain was assessed via activity monitors and Von Frey filament testing. At 4 weeks post-collagenase injections, animals were euthanized., Results: Cage crossings significantly decreased among all rats 2-3 days following each injection period, however, tactile allodynia measures did not reflect this injury response. Biomechanical properties, interleukin-1 beta levels, and glycosaminoglycan content did not differ between groups. While not statistically significant, levels of leukotriene B 4 were lower in the minocycline group compared to controls ( p  = 0.061), suggesting a trend., Conclusions: Our study further characterizes the collagenase model of tendinopathy by demonstrating no evidence of central sensitization with collagenase-induced injury. We found no adverse effect of intratendinous injections of minocycline-loaded poly-lactic-co-glycolic acid microspheres, although no therapeutic effect was observed. Future studies involving a more substantial tendon injury with a greater inflammatory component may be necessary to more thoroughly evaluate the effects of minocycline on tendon pathology., (© 2019 Prof. PK Surendran Memorial Education Foundation. Published by Elsevier, a division of RELX India, Pvt. Ltd. All rights reserved.)