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Mathias K Gehrmann,1 Melanie A Kimm,2 Stefan Stangl,1 Thomas E Schmid,1 Peter B Noël,2 Ernst J Rummeny,2 Gabriele Multhoff11Department of Radiation Oncology, 2Department of Diagnostic and Interventional Radiology, Klinikum rechts der Isar, Technische Universität München, Munich, GermanyAbstract: Real-time imaging of small tumors is still one of the challenges in cancer diagnosis, prognosis, and monitoring of clinical outcome. Targeting novel biomarkers that are selectively expressed on a large variety of different tumors but not normal cells has the potential to improve the imaging capacity of existing methods such as computed tomography. Herein, we present a novel technique using cmHsp70.1 monoclonal antibody-conjugated spherical gold nanoparticles for quantification of the targeted uptake of gold nanoparticles into membrane Hsp70-positive tumor cells. Upon binding, cmHsp70.1-conjugated gold nanoparticles but not nanoparticles coupled to an isotype-matched IgG1 antibody or empty nanoparticles are rapidly taken up by highly malignant Hsp70 membrane-positive mouse tumor cells. After 24 hours, the cmHsp70.1-conjugated gold nanoparticles are found to be enriched in the perinuclear region. Specificity for membrane Hsp70 was shown by using an Hsp70 knockout tumor cell system. Toxic side effects of the cmHsp70.1-conjugated nanoparticles are not observed at a concentration of 1–10 µg/mL. Experiments are ongoing to evaluate whether cmHsp70.1 antibody-conjugated gold nanoparticles are suitable for the detection of membrane-Hsp70-positive tumors in vivo.Keywords: heat shock protein 70, tumor biomarker, theranostics, multimodal CT, multispectral CT, k-edge

Purpose: To assess immunogenic effects in unembolized contralateral tumor after single lobar yttrium-90 transarterial radioembolization ( 90 Y-TARE) of colorectal liver metastases (CRLMs)., Material and Methods: The analysis comprised 10 patients with microsatellite stable (MSS) CRLM scheduled for staged treatment in the prospective AROMA trial. Eligibility criteria included bilobar metastatic disease with >5 lesions without any treatment within 3 weeks. Baseline biopsy was followed by initial 90 Y-TARE treatment of 1 liver lobe, followed by a second biopsy of yet untreated tumors in the other liver lobe at a median of 13 days (range, 4-49 days) immediately before second treatment. Tumor biopsies and peripheral blood mononuclear cells (PBMCs) were collected before treatments for immune cell analysis. Patients were stratified into responders and nonresponders based on tumor control or progression during follow-up., Results: At baseline, responders (n = 4) displayed lower concentrations of FoxP3 + cells and colocation of CD4 + FoxP3 + cells than nonresponders (both P = .02) in tumor tissues. At second biopsy, nonresponders showed a higher CD68 + macrophage density (P = .0014) than responders. Responders displayed fewer CD4 + FoxP3 + T cells than CD8 + T cells at all time points (P = .02 and P = .0428). Nonresponders demonstrated a trending increase in CD68 + macrophages (P = .062), as well as a higher CD8 + PD1 + /CD8 + ratio (P = .062). PBMCs of nonresponders displayed lower CD8 + PD1 + T cells and CD8 + PD1 + /CD8 + ratio at both time points., Conclusions: 90 Y-TARE induces local immunogenic effects in nonexposed MSS CRLM, as well as systemic exhaustion of immune cells in nonresponders. Clinical implications such as a prognostic role or synergism of 90 Y-TARE and checkpoint inhibition in MSS CRLM warrant further investigation., (Copyright © 2024 SIR. Published by Elsevier Inc. All rights reserved.)

Background: Chondrosarcomas are rare malignant bone tumors diagnosed by analyzing radiological images and histology of tissue biopsies and evaluating features such as matrix calcification, cortical destruction, trabecular penetration, and tumor cell entrapment., Methods: We retrospectively analyzed 16 cartilaginous tumor tissue samples from three patients (51-, 54-, and 70-year-old) diagnosed with a dedifferentiated chondrosarcoma at the femur, a moderately differentiated chondrosarcoma in the pelvis, and a predominantly moderately differentiated chondrosarcoma at the scapula, respectively. We combined a hematein-based x-ray staining with high-resolution three-dimensional (3D) microscopic x-ray computed tomography (micro-CT) for nondestructive 3D tumor assessment and tumor margin evaluation., Results: We detected trabecular entrapment on 3D micro-CT images and followed bone destruction throughout the volume. In addition to staining cell nuclei, hematein-based staining also improved the visualization of the tumor matrix, allowing for the distinction between the tumor and the bone marrow cavity. The hematein-based staining did not interfere with further conventional histology. There was a 5.97 ± 7.17% difference between the relative tumor area measured using micro-CT and histopathology (p = 0.806) (Pearson correlation coefficient r = 0.92, p = 0.009). Signal intensity in the tumor matrix (4.85 ± 2.94) was significantly higher in the stained samples compared to the unstained counterparts (1.92 ± 0.11, p = 0.002)., Conclusions: Using nondestructive 3D micro-CT, the simultaneous visualization of radiological and histopathological features is feasible., Relevance Statement: 3D micro-CT data supports modern radiological and histopathological investigations of human bone tumor specimens. It has the potential for being an integrative part of clinical preoperative diagnostics., Key Points: • Matrix calcifications are a relevant diagnostic feature of bone tumors. • Micro-CT detects all clinically diagnostic relevant features of x-ray-stained chondrosarcoma. • Micro-CT has the potential to be an integrative part of clinical diagnostics., (© 2024. The Author(s).)

Background: We aimed to correlate alterations in the rat sarcoma virus (RAS)/mitogen-activated protein kinase pathway in vascular anomalies to the clinical phenotype for improved patient and treatment stratification., Methods and Results: This retrospective multicenter cohort study included 29 patients with extracranial vascular anomalies containing mosaic pathogenic variants (PVs) in genes of the RAS/mitogen-activated protein kinase pathway. Tissue samples were collected during invasive treatment or clinically indicated biopsies. PVs were detected by the targeted sequencing of panels of genes known to be associated with vascular anomalies, performed using DNA from affected tissue. Subgroup analyses were performed according to the affected genes with regard to phenotypic characteristics in a descriptive manner. Twenty-five vascular malformations, 3 vascular tumors, and 1 patient with both a vascular malformation and vascular tumor presented the following distribution of PVs in genes: Kirsten rat sarcoma viral oncogene (n=10), neuroblastoma ras viral oncogene homolog (n=1), Harvey rat sarcoma viral oncogene homolog (n=5), V-Raf murine sarcoma viral oncogene homolog B (n=8), and mitogen-activated protein kinase kinase 1 (n=5). Patients with RAS PVs had advanced disease stages according to the Schobinger classification (stage 3-4: RAS , 9/13 versus non- RAS , 3/11) and more frequent progression after treatment ( RAS , 10/13 versus non- RAS , 2/11). Lesions with Kirsten rat sarcoma viral oncogene PVs infiltrated more tissue layers compared with the other PVs including other RAS PVs (multiple tissue layers: Kirsten rat sarcoma viral oncogene, 8/10 versus other PVs, 6/19)., Conclusions: This comparison of patients with various PVs in genes of the RAS/MAPK pathway provides potential associations with certain morphological and clinical phenotypes. RAS variants were associated with more aggressive phenotypes, generating preliminary data and hypothesis for future larger studies.

Background & Aims: Herein we used data derived from the SORAMIC trial to explore the predictive value of systemic inflammatory markers (neutrophil-to-lymphocyte ratio [NLR] and platelet-to-lymphocyte ratio [PLR]) in patients with advanced hepatocellular carcinoma (HCC) treated with sorafenib monotherapy or the combination of selective internal radiation therapy (SIRT)/sorafenib., Methods: Patients randomized to sorafenib monotherapy or SIRT/sorafenib within the per-protocol population of the SORAMIC trial were evaluated in this exploratory post hoc analysis. The median baseline values of NLR and PLR were used as cut-off values to describe subgroups. Kaplan-Meier curves with log-rank tests were used to evaluate median survival in the sorafenib and SIRT/sorafenib arms in each subgroup. Multivariable Cox regression analysis was applied to eliminate the effect of confounding factors., Results: A total of 275 patients with a median overall survival of 12.4 months were included in this analysis. The median NLR value of the cohort was 2.77 and the median PLR was 26.5. There was no significant difference in overall survival between the sorafenib and SIRT/sorafenib arms in patients with low NLR ( p  = 0.72) and PLR ( p  = 0.35) values. In patients with high NLR values, there was no statistically significant difference in median overall survival between SIRT/sorafenib and sorafenib cohorts (12.1 vs . 9.2 months, p  = 0.21). In patients with high PLR values, overall survival in the SIRT/sorafenib arm was significantly longer than in the sorafenib arm (15.9 vs . 11.0 months, p  = 0.029). This significant difference was preserved in the multivariable analysis (SIRT/sorafenib arm: hazard ratio 0.65, 95% CI 0.44-0.96, p  = 0.03) incorporating age, Child-Pugh grade, and alpha-fetoprotein levels., Conclusions: PLR is a potential predictive factor of benefit from additional SIRT in patients with HCC receiving sorafenib therapy. The potential predictive value of PLR should be further evaluated in future trials., Impact and Implications: Systemic therapies are the mainstay of treatment in patients with hepatocellular carcinoma at advanced stages. However, not all patients respond well to these treatments. In our analysis, using blood test parameters showing systemic inflammation status, we were able to identify patients who would benefit more from combined treatment with a locoregional treatment of radioembolization (or selective internal radiation therapy)., Competing Interests: Osman Öcal: Honoraria: Bayer. Max Seidensticker: Personal fees: Bayer, Sirtex. Najib Ben Khaled: Travel expenses: EISAI. Lecture honoraria: Falk Foundation and Astra Zeneca. Maciej Pech: Grants: Sirtex, Bayer; Personal fees: Sirtex. Bruno Sangro: Consultancy fees: Adaptimmune, Astra Zeneca, Bayer, BMS, Boston Scientific, BTG, Eisai, Eli Lilly, H3 Biomedicine, Ipsen, Novartis, Merck, Roche, Sirtex Medical and Terumo. Speaker fees: Astra Zeneca, Bayer, BMS, BTG, Eli Lilly, Ipsen, Novartis, Merck, Roche, Sirtex Medical and Terumo. Grants (to Institution): BMS and Sirtex Medical. Jens Ricke: Grants: Sirtex, Bayer; Personal fees: Sirtex, Bayer. Moritz Wildgruber: Speaker fees: Sirtex. Please refer to the accompanying ICMJE disclosure forms for further details., (© 2023 The Author(s).)

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 90 Y-radioembolization ( 90 Y-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 90 Y-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 90 Y-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 90 Y-RE showed a trend to shorter OS (p = 0.055)., Conclusion: miR-146a may represent a novel prognostic biomarker for 90 Y-radioembolization in metastatic liver cancer., (© 2023. The Author(s).)

Purpose: Biomarkers are essential to implement personalized therapies in cancer treatment options. As primary liver tumors are increasing and treatment is coupled to liver function and activation of systemic cells of the immune system, we investigated blood-based cells for their ability to predict response to local ablative therapy., Methods: We analyzed peripheral blood cells in 20 patients with primary liver cancer at baseline and following brachytherapy. In addition to platelets, leukocytes, lymphocytes, monocytes, neutrophils and most common ratios PLR, LMR, NMR and NLR, we investigated T cell and NKT cell populations of 11 responders and 9 non-responders using flow cytometry., Results: We have found a peripheral blood cell signature that differed significantly between responders and non-responders treated with interstitial brachytherapy (IBT). At baseline, non-responders featured higher numbers of platelets, monocytes and neutrophils, a higher platelet-to-lymphocyte ratio and an increase in the NKT cell population with a concurrent reduction in CD16 + NKT cells. Simultaneously, a lower percentage of CD4 + T cells was present in non-responders, as also reflected in a lower CD4/8 ratio. CD45RO + memory cells were lower in both, CD4 + and CD8 + T cell populations whereas PD-1 + T cells were only present in the CD4 + T cell population., Conclusion: Baseline blood-based cell signature may function as a biomarker to predict response following brachytherapy in primary liver cancer., (© 2023. The Author(s).)

Purpose: To perform radiofrequency (RF) ablation of hepatocellular carcinoma (HCC) and to assess serological and histopathological markers of tumorigenesis in distant untreated tumors to determine whether these were associated with unfavorable outcomes such as early relapse and increased biological aggressiveness., Materials and Methods: The study cohort comprised 13 patients from a prospective single-arm study. All patients underwent 2 ablation sessions of multifocal HCC nodules 14 days apart. Core biopsy samples of untreated tumors were acquired at baseline and at the time of the second ablation session. Samples were stained immunohistochemically with Ki-67 (proliferation) and CD34 (microvasculature). Blood plasma was obtained at baseline and 2 days after the initial ablation session and analyzed for hepatocyte growth factor (HGF), vascular endothelial growth factor C, and angiopoietin-2 using an enzyme-linked immunosorbent assay. The clinical follow-up period ranged from 7 to 25 months. Patients were stratified as responders (complete remission or limited and delayed recurrence at >6 months; n = 6) or nonresponders (any recurrence within 6 months or >3 new tumors or any new tumor of >3 cm thereafter; n = 7)., Results: In 3 of 7 nonresponders, the Ki-67 index markedly increased in untreated tumors, whereas Ki-67 was stable in all responders. Microvascular density strongly increased in a single nonresponder only. HGF and angiopoietin-2 increased by >30% in 3 of 7 and 4 of 7 nonresponders, respectively, whereas they were stable or decreased in responders. Overall, ≥2 biomarkers were elevated in 6 of 7 (85.7%) nonresponders, whereas 4 of 6 responders demonstrated no increased biomarker and 2 patients demonstrated increase in 1 biomarker only (P = .002)., Conclusions: RF ablation of HCC can produce protumorigenic factors that induce effects in distant untreated tumors. These may potentially function as biomarkers of clinical outcome., (Copyright © 2023 SIR. Published by Elsevier Inc. All rights reserved.)

Purpose: Arteriovenous malformations (AVMs) as rare diseases are diagnostically and therapeutically challenging. Due to the limited evidence regarding treatment outcome, prospective data are needed on how different treatment regimens affect outcome. The aims of this prospective trial are to determine effectiveness, safety, and clinical outcome of multimodal treatment in patients with extracranial AVMs., Materials and Methods: After clinical and magnetic resonance imaging (MRI)-based diagnosis and informed consent, 146 patients (> 4 years and < 70 years) undergoing multimodal therapy in tertiary care vascular anomalies centers will be included in this prospective observational trial. Treatment options include conservative management, medical therapy, minimally invasive image-guided procedures (embolization, sclerotherapy) and surgery as well as combinations of the latter. The primary outcome is the patient-reported QoL 6 months after completion of treatment using the short form-36 health survey version 2 (SF-36v2) and the corresponding short form-10 health survey (SF-10) for children. In addition, clinical presentation (physician-reported signs), MRI imaging (radiological assessment of devascularization), recurrence rate, and therapeutic safety will be analyzed. Further follow-up will be performed after 12, 24, and 36 months. Moreover, liquid biopsies are being obtained from peripheral blood at multiple time points to investigate potential biomarkers for therapy response and disease progression., Discussion: The APOLLON trial is a prospective, multicenter, observational open-label trial with unequal study groups to generate prospective evidence for multimodal treatment of AVMs. A multicenter design with the potential to assess larger populations will provide an increased understanding of multimodal therapy outcome in this orphan disease., Trial Registration: German Clinical Trials Register (identification number: DRKS00021019) https://www.drks.de/drks&#95;web/navigate.do?navigationId=trial.HTML&TRIAL&#95;ID=DRKS00021019 ., (© 2022. The Author(s).)

Histopathology as a diagnostic mainstay for tissue evaluation is strictly a 2D technology. Combining and supplementing this technology with 3D imaging has been proposed as one future avenue towards refining comprehensive tissue analysis. To this end, we have developed a laboratory-based X-ray method allowing for the investigation of tissue samples in three dimensions with isotropic volume information. To assess the potential of our method for micro-morphology evaluation, we selected several kidney regions from three patients with cystic kidney disease, obstructive nephropathy and diabetic glomerulopathy. Tissue specimens were processed using our in-house-developed X-ray eosin stain and investigated with a commercial microCT and our in-house-built NanoCT. The microCT system provided overview scans with voxel sizes of [Formula: see text] and the NanoCT was employed for higher resolutions including voxel sizes from [Formula: see text] to 210 nm. We present a methodology allowing for a precise micro-morphologic investigation in three dimensions which is compatible with conventional histology. Advantages of our methodology are its versatility with respect to multi-scale investigations, being laboratory-based, allowing for non-destructive imaging and providing isotropic volume information. We believe, that after future developmental work this method might contribute to advanced multi-modal tissue diagnostics.

Objective: The objective of this study was to non-invasively differentiate the degree of malignancy in two murine breast cancer models based on identification of distinct tissue characteristics in a metastatic and non-metastatic tumor model using a multiparametric Magnetic Resonance Imaging (MRI) approach., Methods: The highly metastatic 4T1 breast cancer model was compared to the non-metastatic 67NR model. Imaging was conducted on a 9.4 T small animal MRI. The protocol was used to characterize tumors regarding their structural composition, including heterogeneity, intratumoral edema and hemorrhage, as well as endothelial permeability using apparent diffusion coefficient (ADC), T1/T2 mapping and dynamic contrast-enhanced (DCE) imaging. Mice were assessed on either day three, six or nine, with an i.v. injection of the albumin-binding contrast agent gadofosveset. Ex vivo validation of the results was performed with laser ablation-inductively coupled plasma-mass spectrometry (LA-ICP-MS), histology, immunhistochemistry and electron microscopy., Results: Significant differences in tumor composition were observed over time and between 4T1 and 67NR tumors. 4T1 tumors showed distorted blood vessels with a thin endothelial layer, resulting in a slower increase in signal intensity after injection of the contrast agent. Higher permeability was further reflected in higher K trans values, with consecutive retention of gadolinium in the tumor interstitium visible in MRI. 67NR tumors exhibited blood vessels with a thicker and more intact endothelial layer, resulting in higher peak enhancement, as well as higher maximum slope and area under the curve, but also a visible wash-out of the contrast agent and thus lower K trans values. A decreasing accumulation of gadolinium during tumor progression was also visible in both models in LA-ICP-MS. Tissue composition of 4T1 tumors was more heterogeneous, with intratumoral hemorrhage and necrosis and corresponding higher T1 and T2 relaxation times, while 67NR tumors mainly consisted of densely packed tumor cells. Histogram analysis of ADC showed higher values of mean ADC, histogram kurtosis, range and the 90 th percentile (p90), as markers for the heterogenous structural composition of 4T1 tumors. Principal component analysis (PCA) discriminated well between the two tumor models., Conclusions: Multiparametric MRI as presented in this study enables for the estimation of malignant potential in the two studied tumor models via the assessment of certain tumor features over time., Competing Interests: The 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 © 2022 Gerwing, Hoffmann, Kronenberg, Hansen, Masthoff, Helfen, Geyer, Wachsmuth, Höltke, Maus, Hoerr, Krähling, Hiddeßen, Heindel, Karst, Kimm, Schinner, Eisenblätter, Faber and Wildgruber.)

Local ablative therapies are established treatment modalities in the treatment of early- and intermediate-stage hepatocellular carcinoma (HCC). Systemic effects of local ablation on circulating immune cells may contribute to patients' response. Depending on their activation, myeloid cells are able to trigger HCC progression as well as to support anti-tumor immunity. Certain priming of monocytes may already occur while still in the circulation. By using flow cytometry, we analyzed peripheral blood monocyte cell populations from a prospective clinical trial cohort of 21 HCC patients following interstitial brachytherapy (IBT) or radiofrequency ablation (RFA) and investigated alterations in the composition of monocyte subpopulations and monocytic myeloid-derived suppressor cells (mMDSCs) as well as receptors involved in orchestrating monocyte function. We discovered that mMDSC levels increased following both IBT and RFA in virtually all patients. Furthermore, we identified varying alterations in the level of monocyte subpopulations following radiation compared to RFA. (A) Liquid biopsy liquid biopsy of circulating monocytes in the future may provide information on the inflammatory response towards local ablation as part of an orchestrated immune response., Competing Interests: The 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 © 2022 Kimm, Kästle, Stechele, Öcal, Richter, Ümütlü, Schinner, Öcal, Salvermoser, Alunni-Fabbroni, Seidensticker, Goldberg, Ricke and Wildgruber.)

Objective: The aim of this study is to evaluate whether X-ray dark-field (DF) radiography is useful for the diagnosis of gout in birds and reptiles and whether this preclinical model could be helpful to establish this non-invasive imaging method in human medicine., Material and Methods: A total of 18 limbs originating from 11 birds (7 different species) and 7 reptiles (4 different species) with and without suspected joint gout were measured using a grating-based X-ray dark-field setup and conventional X-ray examination, respectively. Each image acquisition generated a dark-field and a conventional absorption x-ray image. The results of the individual scans were compared with the results of a pathological examination and arthrocentesis., Results: In 5 of the birds and 4 of the reptiles examined, gout was detected by pathologic examination. In each group, uric acid crystals were found in the joints of 3 animals by means of arthrocentesis. The uric acid crystals were detectable in 2 bird and 2 reptile limbs in the dark-field image., Conclusion: The study demonstrated that the urate crystals evoke a clearly visible dark field signal, whereas this was not the case in the conventional radiographs., Clinical Relevance: The results obtained show that uric acid crystal detection using less invasive imaging methods in an animal model with birds and reptiles may expand gout diagnostics not only in veterinary medicine but also in human medicine and possibly replace arthrocentesis if a DF signal is detectable. Preclinical scanners which use X-ray dark-field and phase-contrast radiography already exist for hands and mammography., Competing Interests: Die Autoren bestätigen, dass kein Interessenkonflikt besteht., (Thieme. All rights reserved.)

High-Z gold nanoparticles (AuNPs) conjugated to a targeting antibody can help to improve tumor control in radiotherapy while simultaneously minimizing radiotoxicity to adjacent healthy tissue. This paper summarizes the main findings of a joint research program which applied AuNP-conjugates in preclinical modeling of radiotherapy at the Klinikum rechts der Isar, Technical University of Munich and Helmholtz Zentrum München. A pharmacokinetic model of superparamagnetic iron oxide nanoparticles was developed in preparation for a model simulating the uptake and distribution of AuNPs in mice. Multi-scale Monte Carlo simulations were performed on a single AuNP and multiple AuNPs in tumor cells at cellular and molecular levels to determine enhancements in the radiation dose and generation of chemical radicals in close proximity to AuNPs. A biologically based mathematical model was developed to predict the biological response of AuNPs in radiation enhancement. Although simulations of a single AuNP demonstrated a clear dose enhancement, simulations relating to the generation of chemical radicals and the induction of DNA strand breaks induced by multiple AuNPs showed only a minor dose enhancement. The differences in the simulated enhancements at molecular and cellular levels indicate that further investigations are necessary to better understand the impact of the physical, chemical, and biological parameters in preclinical experimental settings prior to a translation of these AuNPs models into targeted cancer radiotherapy.

Inflammation is the phenotypic form of various diseases. Recent development in molecular imaging provides new insights into the diagnostic and therapeutic evaluation of different inflammatory diseases as well as diseases involving inflammation such as cancer. While conventional imaging techniques used in the clinical setting provide only indirect measures of inflammation such as increased perfusion and altered endothelial permeability, optical imaging is able to report molecular information on diseased tissue and cells. Optical imaging is a quick, noninvasive, nonionizing, and easy-to-use diagnostic technology which has been successfully applied for preclinical research. Further development of optical imaging technology such as optoacoustic imaging overcomes the limitations of mere fluorescence imaging, thereby enabling pilot clinical applications in humans. By means of endogenous and exogenous contrast agents, sites of inflammation can be accurately visualized in vivo. This allows for early disease detection and specific disease characterization, enabling more rapid and targeted therapeutic interventions. In this review, we summarize currently available optical imaging techniques used to detect inflammation, including optical coherence tomography (OCT), bioluminescence, fluorescence, optoacoustics, and Raman spectroscopy. We discuss advantages and disadvantages of the different in vivo imaging applications with a special focus on targeting inflammation including immune cell tracking., (© 2021. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Gout is the most common form of inflammatory arthritis, caused by the deposition of monosodium urate (MSU) crystals in peripheral joints and tissue. Detection of MSU crystals is essential for definitive diagnosis, however the gold standard is an invasive process which is rarely utilized. In fact, most patients are diagnosed or even misdiagnosed based on manifested clinical signs, as indicated by the unchanged premature mortality among gout patients over the past decade, although effective treatment is now available. An alternative, non-invasive approach for the detection of MSU crystals is X-ray dark-field radiography. In our work, we demonstrate that dark-field X-ray radiography can detect naturally developed gout in animals with high diagnostic sensitivity and specificity based on the in situ measurement of MSU crystals. With the results of this study as a potential basis for further research, we believe that X-ray dark-field radiography has the potential to substantially improve gout diagnostics., (© 2021. The Author(s).)

The process of restenosis is based on the interplay of various mechanical and biological processes triggered by angioplasty-induced vascular trauma. Early arterial recoil, negative vascular remodeling, and neointimal formation therefore limit the long-term patency of interventional recanalization procedures. The most serious of these processes is neointimal hyperplasia, which can be traced back to 4 main mechanisms: endothelial damage and activation; monocyte accumulation in the subintimal space; fibroblast migration; and the transformation of vascular smooth muscle cells. A wide variety of animal models exists to investigate the underlying pathophysiology. Although mouse models, with their ease of genetic manipulation, enable cell- and molecular-focused fundamental research, and rats provide the opportunity to use stent and balloon models with high throughput, both rodents lack a lipid metabolism comparable to humans. Rabbits instead build a bridge to close the gap between basic and clinical research due to their human-like lipid metabolism, as well as their size being accessible for clinical angioplasty procedures. Every different combination of animal, dietary, and injury model has various advantages and disadvantages, and the decision for a proper model requires awareness of species-specific biological properties reaching from vessel morphology to distinct cellular and molecular features., Competing Interests: Dr Wildgruber was supported by a grant from the Deutsche Forschungsgemeinschaft DFG (WI 3686/7-1)., (© 2021 The Authors.)

Tumor-associated macrophages (TAMs) represent the largest group of leukocytes within the tumor microenvironment (TME) of solid tumors and orchestrate the composition of anti- as well as pro-tumorigenic factors. This makes TAMs an excellent target for novel cancer therapies. The plasticity of TAMs resulting in varying membrane receptors and expression of intracellular proteins allow the specific characterization of different subsets of TAMs. Those markers similarly allow tracking of TAMs by different means of molecular imaging. This review aims to provides an overview of the origin of tumor-associated macrophages, their polarization in different subtypes, and how characteristic markers of the subtypes can be used as targets for molecular imaging and theranostic approaches.

Conventional histology is a destructive technique based on the evaluation of 2D slices of a 3D biopsy. By using 3D X-ray histology these obstacles can be overcome, but their application is still restricted due to the inherently low attenuation properties of soft tissue. In order to solve this problem, the tissue can be stained before X-ray computed tomography imaging (CT) to enhance the soft tissue X-ray contrast. Evaluation of brominated fluorescein salts revealed a mutual influence of the number of bromine atoms and the cations applied on the achieved contrast enhancement. The dibromo fluorescein barium salt turned out to be the ideal X-ray contrast agent, allowing for 3D imaging and subsequent complementing counterstaining applying standard histological techniques., (© 2020 The Authors. Published by Wiley-VCH GmbH.)

Purpose: Myocardial healing following myocardial infarction (MI) is a complex process that is yet to be fully understood. Clinical attempts in regeneration of the injured myocardium using cardiac stem cells faced major challenges, calling for a better understanding of the processes involved at a more basic level in order to foster translation., Procedures: We examined the feasibility of volumetric optoacoustic tomography (VOT) in studying healing of the myocardium in different models of MI, including permanent occlusion (PO) of the left coronary artery, temporary occlusion (ischemia-reperfusion-I/R) and infarcted c-kit mutants, a genetic mouse model with impaired cardiac healing. Murine hearts were imaged at 100 Hz frame rate using 800 nm excitation wavelength, corresponding to the peak absorption of indocyanine green (ICG) in plasma and the isosbestic point of haemoglobin., Results: The non-invasive real-time volumetric imaging capabilities of VOT have allowed the detection of significant variations in the pulmonary transit time (PTT), a parameter affected by MI, across different murine models. Upon intravenous injection of ICG, we were able to track alterations in cardiac perfusion in I/R models, which were absent in wild-type (wt) PO or kit W /kit W-v PO mice. The wt-PO and I/R models further exhibited irregularities in their cardiac cycles., Conclusions: Clear differences in the PTT, ICG perfusion and cardiac cycle patterns were identified between the different models and days post MI. Overall, the results highlight the unique capacity of VOT for multi-parametric characterization of morphological and functional changes in murine models of MI.

Objective: The aim of this study was to evaluate the potential of x-ray dark-field radiography for the noninvasive detection of monosodium urate (MSU) crystals as a novel diagnostic tool for gout., Materials and Methods: Contrast-to-noise ratios of MSU crystals in conventional radiography and dark-field radiography have been compared in a proof of principle measurement. Monosodium urate crystals have been injected into mouse legs in an ex vivo experimental gout setup. Three radiologists independently evaluated the images for the occurrence of crystal deposits in a blinded study for attenuation images only, dark-field images only, and with both images available for a comprehensive diagnosis. All imaging experiments have been performed at an experimental x-ray dark-field setup with a 3-grating interferometer, a rotating anode tube (50 kVp), and a photon-counting detector (effective pixel size, 166 μm)., Results: X-ray dark-field radiography provided a strong signal increase for MSU crystals in a physiological buffer solution compared with conventional attenuation radiography with a contrast-to-noise ratio increase from 0.8 to 19.3. Based on conventional attenuation images only, the reader study revealed insufficient diagnostic performance (sensitivity, 11%; specificity, 92%) with poor interrater agreement (Cohen's coefficient κ = 0.031). Based on dark-field images, the sensitivity increased to 100%, specificity remained at 92%, and the interrater agreement increased to κ = 0.904. Combined diagnosis based on both image modalities maximized both sensitivity and specificity to 100% with absolute interrater agreement (κ = 1.000)., Conclusions: X-ray dark-field radiography enables the detection of MSU crystals in a mouse-based gout model. The simultaneous avaliability of a conventional attenuation image together with the dark-field image provides excellent detection rates of gout deposits with high specificity.

Aims: To correlate signal intensities in grating-based phase-contrast CT (PCCT) images obtained at a synchrotron light source and a conventional X-ray source with tissue components in human liver cirrhosis and hepatocellular carcinoma (HCC) specimen., Methods: Study approval was obtained by the institutional review board. Human specimen of liver cirrhosis and HCC were imaged at experimental grating-based PCCT setups using either a synchrotron radiation source or a conventional X-ray tube. Tissue samples were sectioned and processed for H&E and Elastica van Gieson staining. PCCT and histological images were manually correlated. Depending on morphology and staining characteristics tissue components like fibrosis, HCC, inflammation, connective tissue and necrosis were differentiated and visually correlated with signal intensity in PCCT images using a 5-point Likert scale with normal liver parenchyma as a reference., Results: Grating-based PCCT images of human cirrhotic liver and HCC specimen showed high soft-tissue contrast allowing correlation with histopathological sections. Signal intensities were similar in both setups independent of the nature of the radiation source. Connective tissue and areas of haemorrhage displayed the highest signal intensities, fibrotic liver tissue the lowest., Conclusions: Grating-based PCCT provides comparable results for the characterisation of human specimen of liver cirrhosis and HCC using either a synchrotron light source or a conventional X-ray tube. Due to its high soft-tissue contrast and its applicability to conventional X-ray tubes grating-based PCCT holds potential for preclinical research and virtual histology applications., Competing Interests: Competing interests: PBN reports grants from Philips Healthcare, outside the submitted work., (© Author(s) (or their employer(s)) 2020. No commercial re-use. See rights and permissions. Published by BMJ.)

Imaging techniques such as computed tomographies (CT) play a major role in clinical imaging and diagnosis of malignant lesions. In recent years, metal nanoparticle platforms enabled effective payload delivery for several imaging techniques. Due to the possibility of surface modification, metal nanoparticles are predestined to facilitate molecular tumor targeting. In this work, we demonstrate the feasibility of anti-plasma membrane Heat shock protein 70 (Hsp70) antibody functionalized gold nanoparticles (cmHsp70.1-AuNPs) for tumor-specific multimodal imaging. Membrane-associated Hsp70 is exclusively presented on the plasma membrane of malignant cells of multiple tumor entities but not on corresponding normal cells, predestining this target for a tumor-selective in vivo imaging. In vitro microscopic analysis revealed the presence of cmHsp70.1-AuNPs in the cytosol of tumor cell lines after internalization via the endo-lysosomal pathway. In preclinical models, the biodistribution as well as the intratumoral enrichment of AuNPs were examined 24 h after i.v. injection in tumor-bearing mice. In parallel to spectral CT analysis, histological analysis confirmed the presence of AuNPs within tumor cells. In contrast to control AuNPs, a significant enrichment of cmHsp70.1-AuNPs has been detected selectively inside tumor cells in different tumor mouse models. Furthermore, a machine-learning approach was developed to analyze AuNP accumulations in tumor tissues and organs. In summary, utilizing mHsp70 on tumor cells as a target for the guidance of cmHsp70.1-AuNPs facilitates an enrichment and uniform distribution of nanoparticles in mHsp70-expressing tumor cells that enables various microscopic imaging techniques and spectral-CT-based tumor delineation in vivo.

For fully characterizing renal cell carcinoma (RCC), information about the 3D tissue microstructure is essential. Histopathology, which represents the current diagnostic gold standard, is destructive and only provides 2D information. 3D X-ray histology endeavors to overcome these limitations by generating 3D data. In a laboratory environment, most techniques struggle with limited resolution and the weak X-ray attenuation contrast of soft tissue. We recently developed a laboratory-based method combining nanoscopic X-ray CT with a cytoplasm-specific X-ray stain. Here, we present the application of this method to human RCC biopsies. The NanoCT slices enable pathological characterization of crucial structures by reproducing tissue morphology with a similar detail level as corresponding histological light microscopy images. Beyond that, our data offer deeper insights into the 3D configuration of the tumor. By demonstrating the compatibility of the X-ray stain with standard pathological stains, we highlight the feasibility of integrating staining based NanoCT into the pathological routine.

Most imaging studies of immunotherapy have focused on tracking labeled T cell biodistribution in vivo for understanding trafficking and homing parameters and predicting therapeutic efficacy by the presence of transferred T cells at or in the tumour mass. Conversely, we investigate here a novel concept for longitudinally elucidating anatomical and pathophysiological changes of solid tumours after adoptive T cell transfer in a preclinical set up, using previously unexplored in-tandem macroscopic and mesoscopic optoacoustic (photoacoustic) imaging. We show non-invasive in vivo observations of vessel collapse during tumour rejection across entire tumours and observe for the first time longitudinal tumour rejection in a label-free manner based on optical absorption changes in the tumour mass due to cellular decline. We complement these observations with high resolution episcopic fluorescence imaging of T cell biodistribution using optimized T cell labeling based on two near-infrared dyes targeting the cell membrane and the cytoplasm. We discuss how optoacoustic macroscopy and mesoscopy offer unique contrast and immunotherapy insights, allowing label-free and longitudinal observations of tumour therapy. The results demonstrate optoacoustic imaging as an invaluable tool in understanding and optimizing T cell therapy.

Molecular imaging of atherosclerosis by Magnetic Resonance Imaging (MRI) has been impaired by a lack of validation of the specific substrate responsible for the molecular imaging signal. We therefore aimed to investigate the additive value of mass spectrometry imaging (MSI) of atherosclerosis-affine Gadofluorine P for molecular MRI of atherosclerotic plaques. Atherosclerotic Ldlr -/- mice were investigated by high-field MRI (7 T) at different time points following injection of atherosclerosis-affine Gadofluorine P as well as at different stages of atherosclerosis formation (4, 8, 16 and 20 weeks of HFD). At each imaging time point mice were immediately sacrificed after imaging and aortas were excised for mass spectrometry imaging: Matrix Assisted Laser Desorption Ionization (MALDI) Imaging and Laser Ablation - Inductively Coupled Plasma - Mass Spectrometry (LA-ICP-MS) imaging. Mass spectrometry imaging allowed to visualize the localization and measure the concentration of the MR imaging probe Gadofluorine P in plaque tissue ex vivo with high spatial resolution and thus adds novel and more target specific information to molecular MR imaging of atherosclerosis.

The endothelin (ET) axis plays a pivotal role in cardiovascular diseases. Enhanced levels of circulating ET-1 have been correlated with an inferior clinical outcome after myocardial infarction (MI) in humans. Thus, the evaluation of endothelin-A receptor (ET A R) expression over time in the course of myocardial injury and healing may offer valuable information toward the understanding of the ET axis involvement in MI. We developed an approach to track the expression of ET A R with a customized molecular imaging probe in a murine model of MI. The small molecular probe based on the ET A R-selective antagonist 3-(1,3-benzodioxol-5-yl)-5-hydroxy-5-(4-methoxyphenyl)-4-[(3,4,5-trimethoxyphenyl)methyl]-2(5 H )-furanone (PD156707) was labeled with fluorescent dye, IRDye800cw. Mice undergoing permanent ligation of the left anterior descending artery (LAD) were investigated at day 1, 7, and 21 post surgery after receiving an intravenous injection of the ET A R probe. Cryosections of explanted hearts were analyzed by cryotome-based CCD, and fluorescence reflectance imaging (FRI) and fluorescence signal intensities (SI) were extracted. Fluorescence-mediated tomography (FMT) imaging was performed to visualize probe distribution in the target region in vivo. An enhanced fluorescence signal intensity in the infarct area was detected in cryoCCD images as early as day 1 after surgery and intensified up to 21 days post MI. FRI was capable of detecting significantly enhanced SI in infarcted regions of hearts 7 days after surgery. In vivo imaging by FMT localized enhanced SI in the apex region of infarcted mouse hearts. We verified the localization of the probe and ET A R within the infarct area by immunohistochemistry (IHC). In addition, neovascularized areas were found in the affected myocardium by CD31 staining. Our study demonstrates that the applied fluorescent probe is capable of delineating ET A R expression over time in affected murine myocardium after MI in vivo and ex vivo.

We demonstrate a laboratory-based method combining X-ray microCT and nanoCT with a specific X-ray stain, which targets the cell cytoplasm. The described protocol is easy to apply, fast and suitable for larger soft-tissue samples. The presented methodology enables the characterization of crucial tissue structures in three dimensions and is demonstrated on a whole mouse kidney. The multiscale approach allows to image the entire mouse kidney and supports the selection of further volumes of interest, which are acquired with higher resolutions ranging into the nanometer range. Thereby, soft-tissue morphology with a similar detail level as the corresponding histological light microscopy images is reproduced. Deeper insights into the 3D configuration of tissue structures are achieved without impeding further investigations through histological methods.

Background: Rheumatoid arthritis (RA) is one of the most common rheumatic diseases. Joint inflammation and pathological growth of joint cartilage cause swollen and painful joints, which severely diminishes the patients' life quality. There is no causal treatment. Symptomatic therapies should start as early as possible to take maximal effect. Hence, diagnostic procedures capable of detecting affected joints before the onset of clinical symptoms are highly desirable. We explored the value of PET imaging of integrin subtypes αvβ3 and α5β1 for early detection of RA foci in collagen-induced arthritis (CIA) mouse models., Results: Development of RA in CIA mice was monitored by paw scoring, and αvβ3- and α5β1-integrin expression was quantified by μPET using 68 Ga-Avebetrin and 68 Ga-Aquibeprin. For consecutive sections of selected decalcified joints (knee, ankle), arthritic degeneration and integrin expression were assessed by MOVAT staining and β3/α5 immunohistochemistry (IHC), respectively. β3- and α5-IHC revealed elevated levels of both αvβ3- and α5β1-integrin in arthritic joints. Unlike αvβ3, α5β1 is strongly expressed in the proliferating synovial lining layer, which suggests that its presence is directly related to RA development. For mice with advanced RA (6 weeks after CIA), PET signals for α5β1-integrin were substantially stronger (> 300% of baseline) than that of αvβ3-integrin (< 200%). A longitudinal PET follow-up revealed that the manifestation of clinical symptoms of RA is preceded by upregulation of α5β1- but not of αvβ3-integrin., Conclusion: α5β1-integrin PET could add a new functional imaging aspect to the portfolio of RA diagnostics because it appears to be a sensitive biomarker for early RA development. We suggest α5β1-integrin PET as a valuable tool to achieve a higher precision for early diagnosis of RA, including initial staging, monitoring of the disease course, and drug treatment, and for planning of radiosynoviorthesis (RSO).

Doxorubicin (DOX) is a widely used chemotherapeutic anticancer drug. Its intrinsic fluorescence properties enable investigation of tumor response, drug distribution and metabolism. First phantom studies in vitro showed optoacoustic property of DOX. We therefore aimed to further investigate the optoacoustic properties of DOX in biological tissue in order to explore its potential as theranostic agent. We analysed doxorubicin hydrochloride (Dox·HCl) and liposomal encapsulated doxorubicin hydrochloride (Dox·Lipo), two common drugs for anti-cancer treatment in clinical medicine. Optoacoustic measurements revealed a strong signal of both doxorubicin substrates at 488 nm excitation wavelength. Post mortem analysis of intra-tumoral injections of DOX revealed a detectable optoacoustic signal even at three days after the injection. We thereby demonstrate the general feasibility of doxorubicin detection in biological tissue by means of optoacoustic tomography, which could be applied for high resolution imaging at mesoscopic depths dictated by effective penetration of visible light into the biological tissues., Competing Interests: The authors have declared that no competing interests exist.

Dual-Energy Computed Tomography is of significant clinical interest due to the possibility of material differentiation and quantification. In current clinical routine, primarily two materials are differentiated, e.g., iodine and soft-tissue. A ventilation-perfusion-examination acquired within a single CT scan requires two contrast agents, e.g., xenon and gadolinium, and a three-material differentiation. In the current study, we have developed a solution for three-material differentiation for a ventilation-perfusion-examination. A landrace pig was examined using a dual-layer CT, and three scans were performed: (1) native; (2) xenon ventilation only; (3) xenon ventilation and gadolinium perfusion. An in-house developed algorithm was used to obtain xenon- and gadolinium-density maps. Firstly, lung tissue was segmented from other tissue. Consequently, a two-material decomposition was performed for lung tissue (xenon/soft-tissue) and for remaining tissue (gadolinium/soft-tissue). Results reveal that it was possible to differentiate xenon and gadolinium in a ventilation/perfusion scan of a pig, resulting in xenon and gadolinium density maps. By summation of both density maps, a three-material differentiation (xenon/gadolinium/soft tissue) can be performed and thus, xenon ventilation and gadolinium perfusion can be visualized in a single CT scan. In an additionally performed phantom study, xenon and gadolinium quantification showed very accurate results (r > 0.999 between measured and known concentrations).

The aim was to evaluate the potential of Spectral Photon-Counting Computed Tomography (SPCCT) to differentiate between liquid embolic agents and iodinated contrast medium by using tantalum-characteristic K-edge imaging. Tubes with a concentration series of tantalum and inserts with different concentrations of iodine were scanned with a preclinical SPCCT system. Tantalum density maps (TDM) and iodine density maps (IDM) were generated from a SPCCT acquisition. Furthermore, region-of-interest (ROI) analysis was performed within the tubes in the conventional CT, the TDM and IDM. TDM and IDM enable clear differentiation between both substances. Quantitative measurements of different tantalum concentrations match well with those of actually diluted mixtures. SPCCT allows for differentiation between tantalum and iodine and may enable for an improved follow-up diagnosis in patients after vascular occlusion therapy.

Objective: To investigate whether signal to noise (SNR) analysis of contrast-enhanced MRI gives additional benefit for early disease detection by Magnetic Resonance Imaging (MRI) of experimental rheumatoid arthritis (RA) in a small animal model., Methods: We applied contrast-enhanced MRI at 7T in DBA mice with or without collagen-induced arthritis (CIA). Clinical score, OMERACT RAMRIS analysis and analysis of signal to noise ratios (SNR) of regions of interest in RA bearing mice, methotrexate/methylprednisolone acetate treated RA and control animals were compared with respect to benefit for early diagnosis., Results: While treated RA and control animals did not show signs of RA activity in any of the above-mentioned scoring methods at any time point analyzed, RA animals revealed characteristic signs of RA in RAMRIS at the same time point when RA was detected clinically through scoring of the paws. The MR-based SNR analysis detected signs of synovitis, the earliest indication of RA, not only in late clinical stages, but also at an early stage when little or no clinical signs of RA were present in CIA animals and RAMRIS did not allow a distinct early detection., Conclusion: SNR analysis of contrast-enhanced MR imaging provides additional benefit for early arthritis detection in CIA mice., Competing Interests: The authors have declared that no competing interests exist.

Histological investigations are indispensable with regards to the identification of structural tissue details but are limited to two-dimensional images, which are often visualized in one and the same plane for comparison reasons. Nondestructive three-dimensional technologies such as X-ray micro- and nanoCT have proven to provide valuable benefits for the understanding of anatomical structures as they allow visualization of structural details in 3D and from arbitrary viewing angles. Nevertheless, low attenuation of soft tissue has hampered their application in the field of 3D virtual histology. We present a hematein-based X-ray staining method that specifically targets the cell nuclei of cells, as demonstrated for a whole liver lobule of a mouse. Combining the novel staining protocol with the high resolving power of a recently developed nanoCT system enables the 3D visualization of tissue architecture in the nanometer range, thereby revealing the real 3D morphology and spatial distribution of the cell nuclei. Furthermore, our technique is compatible with conventional histology, as microscopic slides can be derived from the very same stained soft-tissue sample and further counter staining is possible. Thus, our methodology demonstrates future applicability for modern histopathology using laboratory X-ray CT devices.

Objectives: After endovascular aortic repair (EVAR), discrimination of endoleaks and intra-aneurysmatic calcifications within the aneurysm often requires multiphase computed tomography (CT). Spectral photon-counting CT (SPCCT) in combination with a two-contrast agent injection protocol may provide reliable detection of endoleaks with a single CT acquisition., Methods: To evaluate the feasibility of SPCCT, the stent-lined compartment of an abdominal aortic aneurysm phantom was filled with a mixture of iodine and gadolinium mimicking enhanced blood. To represent endoleaks of different flow rates, the adjacent compartments contained either one of the contrast agents or calcium chloride to mimic intra-aneurysmatic calcifications. After data acquisition with a SPCCT prototype scanner with multi-energy bins, material decomposition was performed to generate iodine, gadolinium and calcium maps., Results: In a conventional CT slice, Hounsfield units (HU) of the compartments were similar ranging from 147 to 168 HU. Material-specific maps differentiate the distributions within the compartments filled with iodine, gadolinium or calcium., Conclusion: SPCCT may replace multiphase CT to detect endoleaks without sacrificing diagnostic accuracy. It is a unique feature of our method to capture endoleak dynamics and allow reliable distinction from intra-aneurysmatic calcifications in a single scan, thereby enabling a significant reduction of radiation exposure., Key Points: • SPCCT might enable advanced endoleak detection. • Material maps derived from SPCCT can differentiate iodine, gadolinium and calcium. • SPCCT may potentially reduce radiation burden for EVAR patients under post-interventional surveillance.

Many histological methods require staining of the cytoplasm, which provides instrumental details for diagnosis. One major limitation is the production of 2D images obtained by destructive preparation of 3D tissue samples. X-ray absorption micro- and nanocomputed tomography (microCT and nanoCT) allows for a nondestructive investigation of a 3D tissue sample, and thus aids to determine regions of interest for further histological examinations. However, application of microCT and nanoCT to biological samples (e.g., biopsies) is limited by the missing contrast within soft tissue, which is important to visualize morphological details. We describe an eosin-based preparation overcoming the challenges of contrast enhancement and selectivity for certain tissues. The eosin-based staining protocol is suitable for whole-organ staining, which then enables high-resolution microCT imaging of whole organs and nanoCT imaging of smaller tissue pieces retrieved from the original sample. Our results demonstrate suitability of the eosin-based staining method for diagnostic screening of 3D tissue samples without impeding further diagnostics through histological methods., Competing Interests: The authors declare no conflict of interest.

Chemical staining of soft-tissues can be used as a strategy to increase their low inherent contrast in X-ray absorption micro-computed tomography (micro-CT), allowing to obtain fast three-dimensional structural information of animal organs. Though some staining agents are commonly used in this context, little is known about the staining agents' ability to stain specific types of tissues; the times necessary to provide a sufficient contrast; and the effect of staining solution in distorting the tissue. Here we contribute to studies of animal organs (mouse heart and lungs) using staining combined with dual-energy micro-CT (DECT). DECT was used in order to obtain an additional quantitative measure for the amount of staining agents within the sample in 3D maps. Our results show that the two staining solutions used in this work diffuse differently in the tissues studied, the staining times of some tens of minutes already produce high-quality micro-CT images and, at the concentrations applied in this work, the staining solutions tested do not cause relevant tissue distortions. While one staining solution provides images of the general morphology of the organs, the other reveals organs' features in the order of a hundred micrometers.

Extraction of murine cardiac functional parameters on a beat-by-beat basis is limited with the existing imaging modalities due to insufficient three-dimensional temporal resolution. Faster volumetric imaging methods enabling in vivo characterization of functional parameters are poised to advance cardiovascular research and provide a better understanding of the mechanisms underlying cardiac diseases. We present a new approach based on analyzing contrast-enhanced optoacoustic (OA) images acquired at high volumetric frame rate without using cardiac gating or other approaches for motion correction. We apply an acute murine myocardial infarction model optimized for acquisition of artifact-free optoacoustic imaging data to study cardiovascular hemodynamics. Infarcted hearts (n = 21) could be clearly differentiated from healthy controls (n = 9) based on a significantly higher pulmonary transit time (PTT) (2.25 [2.00-2.41] s versus 1.34 [1.25-1.67] s, p = 0.0235), while no statistically significant difference was observed in the heart rate (318 [252-361] bpm versus 264 [252-320] bpm, p = 0.3129). Nevertheless, nonlinear heartbeat dynamics was stronger in the healthy hearts, as evidenced by the third harmonic component in the heartbeat spectra. MRI data acquired from the same mice further revealed that the PTT increases with the size of infarction and similarly increases with reduced ejection fraction. Moreover, an inverse relationship between infarct PTT and time post-surgery was found, which suggests the occurrence of cardiac healing. In combination with the proven ability of optoacoustics to track targeted probes within the injured myocardium, our method can depict cardiac anatomy, function, and molecular signatures, with both high spatial and temporal resolution. Volumetric four-dimensional optoacoustic characterization of cardiac dynamics with supreme temporal resolution can capture cardiovascular dynamics on a beat-by-beat basis in mouse models of myocardial ischemia., Competing Interests: Competing Interests: The authors have declared that no competing interest exists.

The distribution of intramyocardially injected rabbit MSCs, labeled with the near-infrared dye 1,1'-dioctadecyl-3,3,3',3'-tetramethylindotricarbo-cyanine-iodide (DiR) using hybrid Fluorescence Molecular Tomography-X-ray Computed Tomography (FMT-XCT) and Multispectral Optoacoustic Tomography (MSOT) imaging technologies, was investigated. Viability and induction of apoptosis of DiR labeled MSCs were assessed by XTT- and Caspase-3/-7-testing in vitro . 2 × 10 6 , 2 × 10 5 and 2 × 10 4 MSCs labeled with 5 and 10 μg DiR/ml were injected into fresh frozen rabbit hearts. FMT-XCT, MSOT and fluorescence cryosection imaging were performed. Concentrations up to 10 μg DiR/ml did not cause apoptosis in vitro (p > 0.05). FMT and MSOT imaging of labeled MSCs led to a strong signal. The imaging modalities highlighted a difference in cell distribution and concentration correlated to the number of injected cells. Ex-vivo cryosectioning confirmed the molecular fluorescence signal. FMT and MSOT are sensitive imaging techniques offering high-anatomic resolution in terms of detection and distribution of intramyocardially injected stem cells in a rabbit model.

Objective: Neutrophils accumulate in early atherosclerotic lesions and promote lesion growth. In this study, we evaluated an elastase-specific near-infrared imaging agent for molecular imaging using hybrid fluorescence molecular tomography/x-ray computed tomography., Approach and Results: Murine neutrophils were isolated from bone marrow and incubated with the neutrophil-targeted near-infrared imaging agent Neutrophil Elastase 680 FAST for proof of principle experiments, verifying that the elastase-targeted fluorescent agent is specifically cleaved and activated by neutrophil content after lysis or cell stimulation. For in vivo experiments, low-density lipoprotein receptor-deficient mice were placed on a Western-type diet and imaged after 4, 8, and 12 weeks by fluorescence molecular tomography/x-ray computed tomography. Although this agent remains silent on injection, it produces fluorescent signal after cleavage by neutrophil elastase. After hybrid fluorescence molecular tomography/x-ray computed tomography imaging, mice were euthanized for whole-body cryosectioning and histological analyses. The in vivo fluorescent signal in the area of the aortic arch was highest after 4 weeks of high-fat diet feeding and decreased at 8 and 12 weeks. Ex vivo whole-body cryoslicing confirmed the fluorescent signal to locate to the aortic arch and to originate from the atherosclerotic arterial wall. Histological analysis demonstrated the presence of neutrophils in atherosclerotic lesions., Conclusions: This study provides evidence that elastase-targeted imaging can be used for in vivo detection of early atherosclerosis. This imaging approach may harbor potential in the clinical setting for earlier diagnosis and treatment of atherosclerosis., (© 2016 American Heart Association, Inc.)

Integrins play an important role in tumor progression, invasion and metastasis. Therefore we aimed to evaluate a preclinical imaging approach applying ανβ3 integrin targeted hybrid Fluorescence Molecular Tomography/X-ray Computed Tomography (FMT-XCT) for monitoring tumor progression as well as early therapy response in a syngeneic murine Non-Small Cell Lung Cancer (NSCLC) model. Lewis Lung Carcinomas were grown orthotopically in C57BL/6 J mice and imaged in-vivo using a ανβ3 targeted near-infrared fluorescence (NIRF) probe. ανβ3-targeted FMT-XCT was able to track tumor progression. Cilengitide was able to substantially block the binding of the NIRF probe and suppress the imaging signal. Additionally mice were treated with an established chemotherapy regimen of Cisplatin and Bevacizumab or with a novel MEK inhibitor (Refametinib) for 2 weeks. While μCT revealed only a moderate slowdown of tumor growth, ανβ3 dependent signal decreased significantly compared to non-treated mice already at one week post treatment. ανβ3 targeted imaging might therefore become a promising tool for assessment of early therapy response in the future., (Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.)

Purpose: X-ray phase-contrast imaging (PCI) provides additional information beyond absorption characteristics by detecting the phase shift of the X-ray beam passing through material. The grating-based system works with standard polychromatic X-ray sources, promising a possible clinical implementation. PCI has been shown to provide additional information in soft-tissue samples. The aim of this study was to determine if ex vivo quantitative phase-contrast computed tomography (PCCT) may differentiate between pathologic fluid collections., Materials and Methods: PCCT was performed with the grating interferometry method. A protein serial dilution, human blood samples and 17 clinical samples of pathologic fluid retentions were imaged and correlated with clinical chemistry measurements. Conventional and phase-contrast tomography images were reconstructed. Phase-contrast Hounsfield Units (HUp) were used for quantitative analysis analogously to conventional HU. The imaging was analyzed using overall means, ROI values as well as whole-volume-histograms and vertical gradients. Contrast to noise ratios were calculated between different probes and between imaging methods., Results: HUp showed a very good linear correlation with protein concentration in vitro. In clinical samples, HUp correlated rather well with cell count and triglyceride content. PCI was better than absorption imaging at differentiating protein concentrations in the protein samples as well as at differentiating blood plasma from cellular components. PCI also allowed for differentiation of watery samples (such as lymphoceles) from pus., Conclusion: Phase-contrast computed tomography is a promising tool for the differentiation of pathologic fluids that appear homogenous with conventional attenuation imaging., (Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.)

Low inherent contrast in soft tissues has been limiting the use of X-ray absorption micro-computed tomography (micro-CT) to access high-resolution structural information of animal organs. The staining agents used in micro-CT to improve the contrast fail in providing high-quality images of whole organs of animals due to diffusion problems of the staining agent into the sample. We demonstrate a staining protocol that incorporates a biochemical conditioning step prior to exposure to the staining agent that succeeds in overcoming the diffusion problems, thus quickly providing high-quality micro-CT images of whole organs of mammals. Besides of yielding non-distorted three-dimensional information at the same spatial resolution accessible in histological sections, micro-CT images of whole organs stained by our method enable easy screening of slices along any direction of the volume thus demonstrating new possibilities of structural analysis in biomedical science.

We performed a high-throughput retroviral insertional mutagenesis screen in mouse mammary tumor virus (MMTV)-induced mammary tumors and identified 33 common insertion sites, of which 17 genes were previously not known to be associated with mammary cancer and 13 had not previously been linked to cancer in general. Although members of the Wnt and fibroblast growth factors (Fgf) families were frequently tagged, our exhaustive screening for MMTV insertion sites uncovered a new repertoire of candidate breast cancer oncogenes. We validated one of these genes, Rspo3, as an oncogene by overexpression in a p53-deficient mammary epithelial cell line. The human orthologs of the candidate oncogenes were frequently deregulated in human breast cancers and associated with several tumor parameters. Computational analysis of all MMTV-tagged genes uncovered specific gene families not previously associated with cancer and showed a significant overrepresentation of protein domains and signaling pathways mainly associated with development and growth factor signaling. Comparison of all tagged genes in MMTV and Moloney murine leukemia virus-induced malignancies showed that both viruses target mostly different genes that act predominantly in distinct pathways.