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Purpose: The role of microRNA-146a (miR-146a) in defining the tumor immune microenvironment (TIME) is well established. The aim of this study was to evaluate circulating miR-146a as an early prognostic marker of 90Y-radioembolization (90Y-RE) in metastatic liver cancer and to assess the correlation between circulating miR-146a and TIME cellular composition in distant, yet untreated metastases. Methods: Twenty-one patients with bilobar liver lesions from gastro-intestinal cancer underwent lobar 90Y-RE. Biopsy of contralateral lobe abscopal tumors was acquired at the onset of a second treatment session at a median of 21 days after initial RE, immediately prior to ablation therapy of the contralateral lobe tumor. miR-146a was measured by RT-qPCR in plasma collected 24 h before (T1) and 48 h after (T2) initial unilobar 90Y-RE. The level of miR-146a was correlated with the infiltration of CD4 + , CD8 + , FoxP3 T cells, CD163 + M2 macrophages and immune-exhausted T cells in the abscopal tumor tissue acquired before the second treatment session. Results: Plasma samples collected at T2 showed a higher concentration of miR-146a with respect to T1 in 43% of the patients (p = 0.002). In these patients, tumors revealed a pro-tumorigenic immune composition with enrichment of Tim3 + immune exhausted cells (p = 0.021), in combination with a higher infiltration of CD163 + M2 macrophages and a lower infiltration of CD8 + T cells. Patients with a higher level of miR-146a after 90Y-RE showed a trend to shorter OS (p = 0.055). Conclusion: miR-146a may represent a novel prognostic biomarker for 90Y-radioembolization in metastatic liver cancer. [ABSTRACT FROM AUTHOR]

Background and aims: Prognostic biomarkers identifying patients with early tumor progression after local ablative therapy remain an unmet clinical need. The aim of this study was to investigate circulating miR-21 and miR-210 levels as prognostic biomarkers of HCC treated by CT-guided high-dose rate brachytherapy (HDR-BT). Materials and Methods: 24 consecutive HCC patients (BCLC A and B) treated with CT-guided HDR-BT (1 × 15 Gy) were included in this prospective IRB-approved study. RT-PCR was performed to quantify miR-21 and miR-210 levels in blood samples acquired prior to and 2 d after HDR-BT. Follow-up imaging (contrast-enhanced liver MRI and whole-body CT) was performed in 3 months follow-up intervals. Therapy response was assessed with patients classified as either responders or non-responders (12 each). Responders were defined as having no local or diffuse systemic progression within 6 months and no diffuse systemic progression exceeding 3 nodules/nodule diameter > 3 cm from 6 months to 2 years. Non-responders had recurrence within 6 months and/or tumor progression with > 3 nodules or individual lesion diameter > 3 cm or extrahepatic disease within two years, respectively. Biostatistics included parametric and non-parametric testing (Mann–Whitney-U-test), as well as Kaplan–Meier curve construction. Results: The responder group demonstrated significantly decreasing miR-21 values 2 d post therapy compared to non-responders (median miR-21 2−ΔΔCт: responders 0.73 [IQR 0.34], non-responders 1.53 [IQR 1.48]; p = 0.0102). miR-210 did not show any significant difference between responders and non-responders (median miR-210 2−ΔΔCт: responders 0.74 [IQR 0.45], non-responders 0.99 [IQR 1.13]; p = 0.8399). Kaplan–Meier curves demonstrated significantly shorter time to systemic progression for increased miR-21 (p = 0.0095) but not miR-210 (p = 0.7412), with events accumulating > 1 year post therapy in non-responders (median time to systemic progression 397 days). Conclusion: Increasing circulating miR-21 levels are associated with poor response and shorter time to systemic progression in HDR-BT-treated HCC. This proof-of-concept study provides a basis for further investigation of miR-21 as a prognostic biomarker and potential stratifier in future clinical trials of interventional oncology therapies. Trial registration: In this monocentric clinical study, we analyzed prospectively acquired data of 24 patients from the "ESTIMATE" patient cohort (Studiennummer: DRKS00010587, Deutsches Register Klinischer Studien). Ethical approval was provided by the ethics committee "Ethikkommission bei der LMU München" (reference number "17-346") on June 20, 2017 and August 26, 2020. [ABSTRACT FROM AUTHOR]

Objectives: To investigate contrast-enhanced ultrasound (CEUS) with VEGFR2-targeted microbubbles for monitoring therapy effects of regorafenib on experimental colon carcinomas in rats with correlation to dynamic contrast-enhanced MRI (DCE-MRI) and immunohistochemistry. Materials and Methods: Human colorectal adenocarcinoma xenografts (HT-29) were implanted subcutaneously in n = 21 (n = 11 therapy group; n = 10 control group) female athymic nude rats (Hsd: RH-Foxn1rnu). Animals were imaged at baseline and after a one-week daily treatment with regorafenib or a placebo (10 mg/kg bodyweight), using CEUS with VEGFR2-targeted microbubbles and DCE-MRI. In CEUS tumor perfusion was assessed during an early vascular phase (wash-in area under the curve = WiAUC) and VEGFR2-specific binding during a late molecular phase (signal intensity after 8 (SI8min) and 10 minutes (SI10min)), using a conventional 15L8 linear transducer (transmit frequency 7 MHz, dynamic range 80 dB, depth 25 mm). In DCE-MRI functional parameters plasma flow (PF) and plasma volume (PV) were quantified. For validation purposes, CEUS parameters were correlated with DCE-MRI parameters and immunohistochemical VEGFR2, CD31, Ki-67 and TUNEL stainings. Results: CEUS perfusion parameter WiAUC decreased significantly (116,989 ± 77,048 a.u. to 30,076 ± 27,095a.u.; p = 0.005) under therapy with no significant changes (133,932 ± 65,960 a.u. to 84,316 ± 74,144 a.u.; p = 0.093) in the control group. In the therapy group, the amount of bound microbubbles in the late phase was significantly lower in the therapy than in the control group on day 7 (SI8min: 283 ± 191 vs. 802 ± 460 a.u.; p = 0.006); SI10min: 226 ± 149 vs. 645 ± 461 a.u.; p = 0.009). PF and PV decreased significantly (PF: 147 ± 58 mL/100 mL/min to 71 ± 15 mL/100 mL/min; p = 0.003; PV: 13 ± 3% to 9 ± 4%; p = 0.040) in the therapy group. Immunohistochemistry revealed significantly fewer VEGFR2 (7.2 ± 1.8 vs. 17.8 ± 4.6; p < 0.001), CD31 (8.1 ± 3.0 vs. 20.8 ± 5.7; p < 0.001) and Ki-67 (318.7 ± 94.0 vs. 468.0 ± 133.8; p = 0.004) and significantly more TUNEL (672.7 ± 194.0 vs. 357.6 ± 192.0; p = 0.003) positive cells in the therapy group. CEUS parameters showed significant (p < 0.05) correlations to DCE-MRI parameters and immunohistochemistry. Conclusions: CEUS with VEGFR2-targeted microbubbles allowed for monitoring regorafenib functional and molecular therapy effects on experimental colorectal adenocarcinomas with a significant decline of CEUS and DCE-MRI perfusion parameters as well as a significant reduction of specifically bound microbubbles under therapy, consistent with a reduced expression of VEGFR2. [ABSTRACT FROM AUTHOR]

Background: Molecular changes in HCC development are largely unknown. As the liver plays a fundamental role in the body’s metabolism, metabolic changes are to be expected.We aimed to identify metabolomic changes in HCC in comparison to liver cirrhosis (LC) patients, which could potentially serve as novel biomarkers for HCC diagnosis and prognosis.Metabolite expression from 38 HCC from the SORAMIC trial and 32 LC patients were analyzed by mass spectrometry. Metabolites with significant differences between LC and HCC at baseline were analyzed regarding expression over follow-up. In addition, association with overall survival was tested using univariate Cox proportional-hazard analysis.41 metabolites showed differential expression between LC and HCC patients. 14 metabolites demonstrated significant changes in HCC patients during follow-up. Campesterol, lysophosphatidylcholine, octadecenoic and octadecadienoic acid, and furoylglycine showed a differential expression in the local ablation vs. palliative care group. High expression of eight metabolites (octadecenoic acid, 2-hydroxybutyrate, myo-inositol, isocitrate, erythronic acid, creatinine, pseudouridine, and erythrol) were associated with poor overall survival. The association between poor OS and octadecenoic acid and creatinine remained statistically significant even after adjusting for tumor burden and LC severity.Our findings give promising insides into the metabolic changes during HCC carcinogenesis and provide candidate biomarkers for future studies. Campesterol and furoylglycine in particular were identified as possible biomarkers for HCC progression. Moreover, eight metabolites were detected as predictors for poor overall survival.Aims: Molecular changes in HCC development are largely unknown. As the liver plays a fundamental role in the body’s metabolism, metabolic changes are to be expected.We aimed to identify metabolomic changes in HCC in comparison to liver cirrhosis (LC) patients, which could potentially serve as novel biomarkers for HCC diagnosis and prognosis.Metabolite expression from 38 HCC from the SORAMIC trial and 32 LC patients were analyzed by mass spectrometry. Metabolites with significant differences between LC and HCC at baseline were analyzed regarding expression over follow-up. In addition, association with overall survival was tested using univariate Cox proportional-hazard analysis.41 metabolites showed differential expression between LC and HCC patients. 14 metabolites demonstrated significant changes in HCC patients during follow-up. Campesterol, lysophosphatidylcholine, octadecenoic and octadecadienoic acid, and furoylglycine showed a differential expression in the local ablation vs. palliative care group. High expression of eight metabolites (octadecenoic acid, 2-hydroxybutyrate, myo-inositol, isocitrate, erythronic acid, creatinine, pseudouridine, and erythrol) were associated with poor overall survival. The association between poor OS and octadecenoic acid and creatinine remained statistically significant even after adjusting for tumor burden and LC severity.Our findings give promising insides into the metabolic changes during HCC carcinogenesis and provide candidate biomarkers for future studies. Campesterol and furoylglycine in particular were identified as possible biomarkers for HCC progression. Moreover, eight metabolites were detected as predictors for poor overall survival.Methods: Molecular changes in HCC development are largely unknown. As the liver plays a fundamental role in the body’s metabolism, metabolic changes are to be expected.We aimed to identify metabolomic changes in HCC in comparison to liver cirrhosis (LC) patients, which could potentially serve as novel biomarkers for HCC diagnosis and prognosis.Metabolite expression from 38 HCC from the SORAMIC trial and 32 LC patients were analyzed by mass spectrometry. Metabolites with significant differences between LC and HCC at baseline were analyzed regarding expression over follow-up. In addition, association with overall survival was tested using univariate Cox proportional-hazard analysis.41 metabolites showed differential expression between LC and HCC patients. 14 metabolites demonstrated significant changes in HCC patients during follow-up. Campesterol, lysophosphatidylcholine, octadecenoic and octadecadienoic acid, and furoylglycine showed a differential expression in the local ablation vs. palliative care group. High expression of eight metabolites (octadecenoic acid, 2-hydroxybutyrate, myo-inositol, isocitrate, erythronic acid, creatinine, pseudouridine, and erythrol) were associated with poor overall survival. The association between poor OS and octadecenoic acid and creatinine remained statistically significant even after adjusting for tumor burden and LC severity.Our findings give promising insides into the metabolic changes during HCC carcinogenesis and provide candidate biomarkers for future studies. Campesterol and furoylglycine in particular were identified as possible biomarkers for HCC progression. Moreover, eight metabolites were detected as predictors for poor overall survival.Results: Molecular changes in HCC development are largely unknown. As the liver plays a fundamental role in the body’s metabolism, metabolic changes are to be expected.We aimed to identify metabolomic changes in HCC in comparison to liver cirrhosis (LC) patients, which could potentially serve as novel biomarkers for HCC diagnosis and prognosis.Metabolite expression from 38 HCC from the SORAMIC trial and 32 LC patients were analyzed by mass spectrometry. Metabolites with significant differences between LC and HCC at baseline were analyzed regarding expression over follow-up. In addition, association with overall survival was tested using univariate Cox proportional-hazard analysis.41 metabolites showed differential expression between LC and HCC patients. 14 metabolites demonstrated significant changes in HCC patients during follow-up. Campesterol, lysophosphatidylcholine, octadecenoic and octadecadienoic acid, and furoylglycine showed a differential expression in the local ablation vs. palliative care group. High expression of eight metabolites (octadecenoic acid, 2-hydroxybutyrate, myo-inositol, isocitrate, erythronic acid, creatinine, pseudouridine, and erythrol) were associated with poor overall survival. The association between poor OS and octadecenoic acid and creatinine remained statistically significant even after adjusting for tumor burden and LC severity.Our findings give promising insides into the metabolic changes during HCC carcinogenesis and provide candidate biomarkers for future studies. Campesterol and furoylglycine in particular were identified as possible biomarkers for HCC progression. Moreover, eight metabolites were detected as predictors for poor overall survival.Conclusion: Molecular changes in HCC development are largely unknown. As the liver plays a fundamental role in the body’s metabolism, metabolic changes are to be expected.We aimed to identify metabolomic changes in HCC in comparison to liver cirrhosis (LC) patients, which could potentially serve as novel biomarkers for HCC diagnosis and prognosis.Metabolite expression from 38 HCC from the SORAMIC trial and 32 LC patients were analyzed by mass spectrometry. Metabolites with significant differences between LC and HCC at baseline were analyzed regarding expression over follow-up. In addition, association with overall survival was tested using univariate Cox proportional-hazard analysis.41 metabolites showed differential expression between LC and HCC patients. 14 metabolites demonstrated significant changes in HCC patients during follow-up. Campesterol, lysophosphatidylcholine, octadecenoic and octadecadienoic acid, and furoylglycine showed a differential expression in the local ablation vs. palliative care group. High expression of eight metabolites (octadecenoic acid, 2-hydroxybutyrate, myo-inositol, isocitrate, erythronic acid, creatinine, pseudouridine, and erythrol) were associated with poor overall survival. The association between poor OS and octadecenoic acid and creatinine remained statistically significant even after adjusting for tumor burden and LC severity.Our findings give promising insides into the metabolic changes during HCC carcinogenesis and provide candidate biomarkers for future studies. Campesterol and furoylglycine in particular were identified as possible biomarkers for HCC progression. Moreover, eight metabolites were detected as predictors for poor overall survival.Graphical Abstract: Molecular changes in HCC development are largely unknown. As the liver plays a fundamental role in the body’s metabolism, metabolic changes are to be expected.We aimed to identify metabolomic changes in HCC in comparison to liver cirrhosis (LC) patients, which could potentially serve as novel biomarkers for HCC diagnosis and prognosis.Metabolite expression from 38 HCC from the SORAMIC trial and 32 LC patients were analyzed by mass spectrometry. Metabolites with significant differences between LC and HCC at baseline were analyzed regarding expression over follow-up. In addition, association with overall survival was tested using univariate Cox proportional-hazard analysis.41 metabolites showed differential expression between LC and HCC patients. 14 metabolites demonstrated significant changes in HCC patients during follow-up. Campesterol, lysophosphatidylcholine, octadecenoic and octadecadienoic acid, and furoylglycine showed a differential expression in the local ablation vs. palliative care group. High expression of eight metabolites (octadecenoic acid, 2-hydroxybutyrate, myo-inositol, isocitrate, erythronic acid, creatinine, pseudouridine, and erythrol) were associated with poor overall survival. The association between poor OS and octadecenoic acid and creatinine remained statistically significant even after adjusting for tumor burden and LC severity.Our findings give promising insides into the metabolic changes during HCC carcinogenesis and provide candidate biomarkers for future studies. Campesterol and furoylglycine in particular were identified as possible biomarkers for HCC progression. Moreover, eight metabolites were detected as predictors for poor overall survival. [ABSTRACT FROM AUTHOR]

Purpose: To investigate αvβ3-integrin-targeted optoacoustic imaging and MRI for monitoring a BRAF/MEK inhibitor combination therapy in a murine model of human melanoma. Materials and methods: Human BRAF V600E-positive melanoma xenograft (A375)-bearing Balb/c nude mice (n = 10) were imaged before (day 0) and after (day 7) a BRAF/MEK inhibitor combination therapy (encorafenib, 1.3 mg/kg/d; binimetinib, 0.6 mg/kg/d, n = 5) or placebo (n = 5), respectively. Optoacoustic imaging was performed on a preclinical system unenhanced and 5 h after i. v. injection of an αvβ3-integrin-targeted fluorescent probe. The αvβ3-integrin-specific tumor signal was derived by spectral unmixing. For morphology-based tumor response assessments, T2w MRI data sets were acquired on a clinical 3 Tesla scanner. The imaging results were validated by multiparametric immunohistochemistry (ß3 –integrin expression, CD31 –microvascular density, Ki-67 –proliferation). Results: The αvβ3-integrin-specific tumor signal was significantly reduced under therapy, showing a unidirectional decline in all animals (from 7.98±2.22 to 1.67±1.30; p = 0.043). No significant signal change was observed in the control group (from 6.60±6.51 to 3.67±1.93; p = 0.500). Immunohistochemistry revealed a significantly lower integrin expression (ß3: 0.20±0.02 vs. 0.39±0.05; p = 0.008) and microvascular density (CD31: 119±15 vs. 292±49; p = 0.008) in the therapy group. Tumor volumes increased with no significant intergroup difference (therapy: +107±42 mm3; control +112±44mm3, p = 0.841). In vivo blocking studies with αvβ3-integrin antagonist cilengitide confirmed the target specificity of the fluorescent probe. Conclusions: αvβ3-integrin-targeted optoacoustic imaging allowed for the early non-invasive monitoring of a BRAF/MEK inhibitor combination therapy in a murine model of human melanoma, adding molecular information on tumor receptor status to morphology-based tumor response criteria. [ABSTRACT FROM AUTHOR]

Objectives: To investigate 68Ga-TRAP-(RGD)3 hybrid imaging for the in vivo monitoring of αvß3-integrin expression as biomarker of anti-angiogenic therapy effects in experimental breast cancer. Materials and Methods: Human breast cancer (MDA-MB-231) xenografts were implanted orthotopically into the mammary fat pads of n = 25 SCID mice. Transmission/emission scans (53 min to 90 min after i.v. injection of 20 MBq 68Ga-TRAP-(RGD)3) were performed on a dedicated small animal PET before (day 0, baseline) and after (day 7, follow-up) a 1-week therapy with the VEGF antibody bevacizumab or placebo (imaging cohort n = 13; therapy n = 7, control n = 6). The target-to-background ratio (TBR, VOImaxtumor/VOImeanmuscle) served as semiquantitative measure of tumor radiotracer uptake. Unenhanced CT data sets were subsequently acquired for anatomic coregistration and morphology-based tumor response assessments (CT volumetry). The imaging results were validated by multiparametric ex vivo immunohistochemistry (αvß3-integrin, microvascular density–CD31, proliferation–Ki-67, apoptosis–TUNEL) conducted in a dedicated immunohistochemistry cohort (n = 12). Results: 68Ga-TRAP-(RGD)3 binding was significantly reduced under VEGF inhibition and decreased in all bevacizumab-treated animals (ΔTBRfollow-up/baseline: therapy -1.07±0.83, control +0.32±1.01, p = 0.022). No intergroup difference in tumor volume development between day 0 and day 7 was observed (Δvolumetherapy 134±77 μL, Δvolumecontrol 132±56 μL, p = 1.000). Immunohistochemistry revealed a significant reduction of αvß3-integrin expression (308±135 vs. 635±325, p = 0.03), microvascular density (CD31, 168±108 vs. 432±70, p = 0.002), proliferation (Ki-67, 5,195±1,002 vs. 7,574±418, p = 0.004) and significantly higher apoptosis (TUNEL, 14,432±1,974 vs. 3,776±1,378, p = 0.002) in the therapy compared to the control group. Conclusions: 68Ga-TRAP-(RGD)3 hybrid imaging allows for the in vivo assessment of αvß3-integrin expression as biomarker of anti-angiogenic therapy effects in experimental breast cancer. [ABSTRACT FROM AUTHOR]

Objective: To investigate annexin-based optical fluorescence imaging (OI) for monitoring regorafenib-induced early cell death in experimental colon carcinomas in rats, validated by perfusion MRI and multiparametric immunohistochemistry. Materials and Methods: Subcutaneous human colon carcinomas (HT-29) in athymic rats (n = 16) were imaged before and after a one-week therapy with regorafenib (n = 8) or placebo (n = 8) using annexin-based OI and perfusion MRI at 3 Tesla. Optical signal-to-noise ratio (SNR) and MRI tumor perfusion parameters (plasma flow PF, mL/100mL/min; plasma volume PV, %) were assessed. On day 7, tumors underwent immunohistochemical analysis for tumor cell apoptosis (TUNEL), proliferation (Ki-67), and microvascular density (CD31). Results: Apoptosis-targeted OI demonstrated a tumor-specific probe accumulation with a significant increase of tumor SNR under therapy (mean Δ +7.78±2.95, control: -0.80±2.48, p = 0.021). MRI detected a significant reduction of tumor perfusion in the therapy group (mean ΔPF -8.17±2.32 mL/100 mL/min, control -0.11±3.36 mL/100 mL/min, p = 0.036). Immunohistochemistry showed significantly more apoptosis (TUNEL; 11392±1486 vs. 2921±334, p = 0.001), significantly less proliferation (Ki-67; 1754±184 vs. 2883±323, p = 0.012), and significantly lower microvascular density (CD31; 107±10 vs. 182±22, p = 0.006) in the therapy group. Conclusions: Annexin-based OI allowed for the non-invasive monitoring of regorafenib-induced early cell death in experimental colon carcinomas, validated by perfusion MRI and multiparametric immunohistochemistry. [ABSTRACT FROM AUTHOR]

Objectives: To investigate a multimodal, multiparametric perfusion MRI / 18F-fluoro-deoxyglucose-(18F-FDG)-PET imaging protocol for monitoring regorafenib therapy effects on experimental colorectal adenocarcinomas in rats with immunohistochemical validation. Materials and Methods: Human colorectal adenocarcinoma xenografts (HT-29) were implanted subcutaneously in n = 17 (n = 10 therapy group; n = 7 control group) female athymic nude rats (Hsd:RH-Foxn1rnu). Animals were imaged at baseline and after a one-week daily treatment protocol with regorafenib (10 mg/kg bodyweight) using a multimodal, multiparametric perfusion MRI/18F-FDG-PET imaging protocol. In perfusion MRI, quantitative parameters of plasma flow (PF, mL/100 mL/min), plasma volume (PV, %) and endothelial permeability-surface area product (PS, mL/100 mL/min) were calculated. In 18F-FDG-PET, tumor-to-background-ratio (TTB) was calculated. Perfusion MRI parameters were correlated with TTB and immunohistochemical assessments of tumor microvascular density (CD-31) and cell proliferation (Ki-67). Results: Regorafenib significantly (p<0.01) suppressed PF (81.1±7.5 to 50.6±16.0 mL/100mL/min), PV (12.1±3.6 to 7.5±1.6%) and PS (13.6±3.2 to 7.9±2.3 mL/100mL/min) as well as TTB (3.4±0.6 to 1.9±1.1) between baseline and day 7. Immunohistochemistry revealed significantly (p<0.03) lower tumor microvascular density (CD-31, 7.0±2.4 vs. 16.1±5.9) and tumor cell proliferation (Ki-67, 434.0 ± 62.9 vs. 663.0 ± 98.3) in the therapy group. Perfusion MRI parameters ΔPF, ΔPV and ΔPS showed strong and significant (r = 0.67-0.78; p<0.01) correlations to the PET parameter ΔTTB and significant correlations (r = 0.57-0.67; p<0.03) to immunohistochemical Ki-67 as well as to CD-31-stainings (r = 0.49-0.55; p<0.05). Conclusions: A multimodal, multiparametric perfusion MRI/PET imaging protocol allowed for non-invasive monitoring of regorafenib therapy effects on experimental colorectal adenocarcinomas in vivo with significant correlations between perfusion MRI parameters and 18F-FDG-PET validated by immunohistochemistry. [ABSTRACT FROM AUTHOR]

Simple Summary: This study aims at using a multi-technique approach to detect and analyze the effects of high dose rate spatially fractionated radiation therapies and to compare them to seamless broad beam irradiation targeting healthy and glioblastoma-bearing rat brains and delivering three different doses per each irradiation geometry. Brains were analyzed post mortem by multi-scale X-ray phase contrast imaging–computed tomography, histology, immunohistochemistry, X-ray fluorescence, and small- and wide-angle X-ray scattering to achieve detailed visualization, characterization and classification in 3D of the radio-induced effects on brain tissues. The original results bring new insights to the understanding of the response of cerebral tissue and tumors treated with high dose rate spatially fractioned radiotherapies and put the basis for channeling studies of in-vivo applications for monitoring RT effects. The purpose of this study is to use a multi-technique approach to detect the effects of spatially fractionated X-ray Microbeam (MRT) and Minibeam Radiation Therapy (MB) and to compare them to seamless Broad Beam (BB) irradiation. Healthy- and Glioblastoma (GBM)-bearing male Fischer rats were irradiated in-vivo on the right brain hemisphere with MRT, MB and BB delivering three different doses for each irradiation geometry. Brains were analyzed post mortem by multi-scale X-ray Phase Contrast Imaging–Computed Tomography (XPCI-CT), histology, immunohistochemistry, X-ray Fluorescence (XRF), Small- and Wide-Angle X-ray Scattering (SAXS/WAXS). XPCI-CT discriminates with high sensitivity the effects of MRT, MB and BB irradiations on both healthy and GBM-bearing brains producing a first-time 3D visualization and morphological analysis of the radio-induced lesions, MRT and MB induced tissue ablations, the presence of hyperdense deposits within specific areas of the brain and tumor evolution or regression with respect to the evaluation made few days post-irradiation with an in-vivo magnetic resonance imaging session. Histology, immunohistochemistry, SAXS/WAXS and XRF allowed identification and classification of these deposits as hydroxyapatite crystals with the coexistence of Ca, P and Fe mineralization, and the multi-technique approach enabled the realization, for the first time, of the map of the differential radiosensitivity of the different brain areas treated with MRT and MB. 3D XPCI-CT datasets enabled also the quantification of tumor volumes and Ca/Fe deposits and their full-organ visualization. The multi-scale and multi-technique approach enabled a detailed visualization and classification in 3D of the radio-induced effects on brain tissues bringing new essential information towards the clinical implementation of the MRT and MB radiation therapy techniques. [ABSTRACT FROM AUTHOR]