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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.

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.

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 multicontrast 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. [ABSTRACT FROM AUTHOR]

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 pO2 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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]