A surgical strategy for intrahepatic cholangiocarcinoma - the hilar first concept.

PURPOSE: The present study assesses long-term overall survival (OS) and disease-free survival (DFS) after curative resection for intrahepatic cholangiocarcinoma (ICCA) depending on resection margin (RM) status and lymph node (LN) status. METHODS: Clinical data of all consecutively resected patients with ICCA at a single high-volume center between 2005 and 2018 were collected. Minimum follow-up was 36 months. Perioperative and long-term oncological outcome was assessed. RESULTS: One hundred ninety-two cases were included in the analysis. Thirty- and 90-day-mortality was 5.2% (n = 10) and 10.9% (n = 21). OS was 26 months with 1-, 2-, and 5-year-OS rates of 72%, 53%, and 26%. One-, 2-, and 5-year-DFS rates were 54%, 42%, and 35% (N0 vs. N1: 29 vs. 9 months, p = 0.116). R1 was not found to be an independent risk factor for reduced survival in the overall cohort (p = 0.098). When differentiating according to the LN status, clear resection margins were significantly associated with increased DFS for N0 cases (50 months vs. 9 months, p = 0.004). For N1 cases, no significant difference in DFS was calculated for R0 compared to R1 cases (9 months vs. 9 months, p = 0.88). For N0 cases, clear resection margins > 10 mm were associated with prolonged OS (p = 0.048). CONCLUSION: For N1 cases, there was no significant survival benefit when comparing R0 versus R1, while the complication rate remained high for the extended resection types. In view of merging multimodal treatment, the hilar first concept assesses locoregional LN status for optimal surgical therapy.

A Cellular Ground Truth to Develop MRI Signatures in Glioma Models by Correlative Light Sheet Microscopy and Atlas-Based Coregistration.

Glioblastoma is the most common malignant primary brain tumor with poor overall survival. Magnetic resonance imaging (MRI) is the main imaging modality for glioblastoma but has inherent shortcomings. The molecular and cellular basis of MR signals is incompletely understood. We established a ground truth-based image analysis platform to coregister MRI and light sheet microscopy (LSM) data to each other and to an anatomic reference atlas for quantification of 20 predefined anatomic subregions. Our pipeline also includes a segmentation and quantification approach for single myeloid cells in entire LSM datasets. This method was applied to three preclinical glioma models in male and female mice (GL261, U87MG, and S24), which exhibit different key features of the human glioma. Multiparametric MR data including T2-weighted sequences, diffusion tensor imaging, T2 and T2* relaxometry were acquired. Following tissue clearing, LSM focused on the analysis of tumor cell density, microvasculature, and innate immune cell infiltration. Correlated analysis revealed differences in quantitative MRI metrics between the tumor-bearing and the contralateral hemisphere. LSM identified tumor subregions that differed in their MRI characteristics, indicating tumor heterogeneity. Interestingly, MRI signatures, defined as unique combinations of different MRI parameters, differed greatly between the models. The direct correlation of MRI and LSM allows an in-depth characterization of preclinical glioma and can be used to decipher the structural, cellular, and, likely, molecular basis of tumoral MRI biomarkers. Our approach may be applied in other preclinical brain tumor or neurologic disease models, and the derived MRI signatures could ultimately inform image interpretation in a clinical setting.SIGNIFICANCE STATEMENT We established a histologic ground truth-based approach for MR image analyses and tested this method in three preclinical glioma models exhibiting different features of glioblastoma. Coregistration of light sheet microscopy to MRI allowed for an evaluation of quantitative MRI data in histologically distinct tumor subregions. Coregistration to a mouse brain atlas enabled a regional comparison of MRI parameters with a histologically informed interpretation of the results. Our approach is transferable to other preclinical models of brain tumors and further neurologic disorders. The method can be used to decipher the structural, cellular, and molecular basis of MRI signal characteristics. Ultimately, information derived from such analyses could strengthen the neuroradiological evaluation of glioblastoma as they enhance the interpretation of MRI data.

Brain tumor classification of virtual NMR voxels based on realistic blood vessel-induced spin dephasing using support vector machines.

Remodeling of tissue microvasculature commonly promotes neoplastic growth; however, there is no imaging modality in oncology yet that noninvasively quantifies microvascular changes in clinical routine. Although blood capillaries cannot be resolved in typical magnetic resonance imaging (MRI) measurements, their geometry and distribution influence the integral nuclear magnetic resonance (NMR) signal from each macroscopic MRI voxel. We have numerically simulated the expected transverse relaxation in NMR voxels with different dimensions based on the realistic microvasculature in healthy and tumor-bearing mouse brains (U87 and GL261 glioblastoma). The 3D capillary structure in entire, undissected brains was acquired using light sheet fluorescence microscopy to produce large datasets of the highly resolved cerebrovasculature. Using this data, we trained support vector machines to classify virtual NMR voxels with different dimensions based on the simulated spin dephasing accountable to field inhomogeneities caused by the underlying vasculature. In prediction tests with previously blinded virtual voxels from healthy brain tissue and GL261 tumors, stable classification accuracies above 95% were reached. Our results indicate that high classification accuracies can be stably attained with achievable training set sizes and that larger MRI voxels facilitated increasingly successful classifications, even with small training datasets. We were able to prove that, theoretically, the transverse relaxation process can be harnessed to learn endogenous contrasts for single voxel tissue type classifications on tailored MRI acquisitions. If translatable to experimental MRI, this may augment diagnostic imaging in oncology with automated voxel-by-voxel signal interpretation to detect vascular pathologies.

Large-scale characterization of the microvascular geometry in development and disease by tissue clearing and quantitative ultramicroscopy.

Three-dimensional assessment of optically cleared, entire organs and organisms has recently become possible by tissue clearing and selective plane illumination microscopy ("ultramicroscopy"). Resulting datasets can be highly complex, encompass over a thousand images with millions of objects and data of several gigabytes per acquisition. This constitutes a major challenge for quantitative analysis. We have developed post-processing tools to quantify millions of microvessels and their distribution in three-dimensional datasets from ultramicroscopy and demonstrate the capabilities of our pipeline within entire mouse brains and embryos. Using our developed acquisition, segmentation, and analysis platform, we quantify physiological vascular networks in development and the healthy brain. We compare various geometric vessel parameters (e.g. vessel density, radius, tortuosity) in the embryonic spinal cord and brain as well as in different brain regions (basal ganglia, corpus callosum, cortex). White matter tract structures (corpus callosum, spinal cord) showed lower microvascular branch densities and longer vessel branch length compared to grey matter (cortex, basal ganglia). Furthermore, we assess tumor neoangiogenesis in a mouse glioma model to compare tumor core and tumor border. The developed methodology allows rapid quantification of three-dimensional datasets by semi-automated segmentation of fluorescently labeled objects with conventional computer hardware. Our approach can aid preclinical investigations and paves the way towards "quantitative ultramicroscopy".

Influence of the amount of intermetallics on the degradation of Mg-Nd alloys under physiological conditions.

The influence of amount of intermetallics on the degradation of as-extruded Mg-Nd alloys with different contents of Nd was investigated via immersion testing in DMEM+10% FBS under cell culture conditions and subsequent microstructural characterizations. It is found that the presence of intermetallic particles Mg41Nd5 affects the corrosion of Mg-Nd alloys in two conflicting ways. One is their negative role that their existence enhances the micro-galvanic corrosion. Another is their positive role. Their existence favours the formation of a continuous and compact corrosion layer. At the early stage of immersion, their negative role predominated. The degradation rate of Mg-Nd alloys monotonously increases with increasing the amount of intermetallics. Mg-5Nd alloy with maximum amount of intermetallics suffered from the most severe corrosion. With the immersion proceeding (≥7 days), then the positive role of these intermetallic particles Mg41Nd5 could not be neglected. Owing to the interaction between their positive and negative roles, at the later stage of immersion the corrosion rate of Mg-Nd alloys first increases with increasing the content of Nd, then reaches to the maximum at 2 wt. % Nd. With a further increase of Nd content, a decrease in corrosion rate occurs. The main corrosion products on the surfaces of Mg-Nd alloys include carbonates, calcium-phosphate, neodymium oxide and/or neodymium hydroxide. They are amorphous at the early stage of immersion. With the immersion proceeding, they are transformed to crystalline. The existence of undegradable Mg41Nd5 particles in the corrosion layer can enhance the crystallization of such amorphous corrosion products.

Thyroid surgery in children and young adults: potential overtreatment and complications.

PURPOSE: Thyroid nodules in the pediatric population are more frequently associated with malignant thyroid disease than in adult cohorts. Yet, there is a potential risk of surgical overtreatment. With this single center study, an analysis of potential overtreatment for suspected malignant thyroid disease in children and young adults was aimed for. METHODS: In a period from 2005 to 2018, 155 thyroid operations in children and young adults performed at the University Medical Center Mainz, Germany, were analyzed (patient age 3-20 years, 117 female). Cases were categorized for preoperative diagnosis: non-malignant (group I, n = 45) and malignant thyroid disease (group II, n = 110). Postoperative parameters (histology, complication rates) were assessed and compared between groups. RESULTS: 91.1% of group I were histologically benign. 44.5% of group II harbored malignancy. Permanent hypoparathyroidism was documented in group I (2.7%) and in group II (1.4%, p = 1.000). Wound infections were absent in group I but observed in group II (0.9%, p = 1.000). Transient vocal cord palsy was recorded only in group I (2.3%, 2/85 vs. 0/177 nerves at risk, p = 0.104). Permanent vocal cord palsies were absent. CONCLUSION: Preoperative diagnoses were correct in over 90% of group I and in nearly 45% of group II. The high proportion of carcinomas in group II ruled out the issue of potential overtreatment. The risk of severe postoperative complications was equally low in both patient groups.

Cardiac iron concentration in relation to systemic iron status and disease severity in non-ischaemic heart failure with reduced ejection fraction.

AIMS: Low cardiac iron levels promote heart failure in experimental models. While cardiac iron concentration (CI) is decreased in patients with advanced heart failure with reduced ejection fraction (HFrEF), CI has never been measured in non-advanced HFrEF. We measured CI in left ventricular (LV) endomyocardial biopsies (EMB) from patients with non-advanced HFrEF and explored CI association with systemic iron status and disease severity. METHODS AND RESULTS: We enrolled 80 consecutive patients with non-ischaemic HFrEF with New York Heart Association class II or III symptoms and a median (interquartile range) LV ejection fraction of 25 (18-33)%. CI was 304 (262-373) µg/g dry tissue. CI was not related to immunohistological findings or the presence of cardiotropic viral genomes in EMBs and was not related to biomarkers of systemic iron status or anaemia. Patients with CI in the lowest quartile (CIQ1 ) had lower body mass indices and more often presented with heart failure histories longer than 6 months than patients in the upper three quartiles (CIQ2-4 ). CIQ1 patients had higher serum N-terminal pro-B-type natriuretic peptide levels than CIQ2-4 patients [3566 (1513-6412) vs. 1542 (526-2811) ng/L; P = 0.005]. CIQ1 patients also had greater LV end-diastolic (P = 0.001) and end-systolic diameter indices (P = 0.003) and higher LV end-diastolic pressures (P = 0.046) than CIQ2-4 patients. CONCLUSION: Low CI is associated with greater disease severity in patients with non-advanced non-ischaemic HFrEF. CI is unrelated to systemic iron homeostasis. The prognostic and therapeutic implications of CI measurements in EMBs should be further explored.

Gibbs point field model quantifies disorder in microvasculature of U87-glioblastoma.

Microvascular proliferation in glioblastoma multiforme is a biological key mechanism to facilitate tumor growth and infiltration and a main target for treatment interventions. The vascular architecture can be obtained by Single Plane Illumination Microscopy (SPIM) to evaluate vascular heterogeneity in tumorous tissue. We make use of the Gibbs point field model to quantify the order of regularity in capillary distributions found in the U87 glioblastoma model in a murine model and to compare tumorous and healthy brain tissue. A single model parameter Γ was assigned that is linked to tissue-specific vascular topology through Monte-Carlo simulations. Distributions of the model parameter Γ differ significantly between glioblastoma tissue with mean 〈ΓG〉=2.1±0.4, as compared to healthy brain tissue with mean 〈ΓH〉=4.9±0.4, suggesting that the average Γ-value allows for tissue differentiation. These results may be used for diagnostic magnetic resonance imaging, where it has been shown recently that Γ is linked to tissue-inherent relaxation parameters.

PERK-mediated expression of peptidylglycine α-amidating monooxygenase supports angiogenesis in glioblastoma.

PKR-like kinase (PERK) plays a significant role in inducing angiogenesis in various cancer types including glioblastoma. By proteomics analysis of the conditioned medium from a glioblastoma cell line treated with a PERK inhibitor, we showed that peptidylglycine α-amidating monooxygenase (PAM) expression is regulated by PERK under hypoxic conditions. Moreover, PERK activation via CCT020312 (a PERK selective activator) increased the cleavage and thus the generation of PAM cleaved cytosolic domain (PAM sfCD) that acts as a signaling molecule from the cytoplasm to the nuclei. PERK was also found to interact with PAM, suggesting a possible involvement in the generation of PAM sfCD. Knockdown of PERK or PAM reduced the formation of tubes by HUVECs in vitro. Furthermore, in vivo data highlighted the importance of PAM in the growth of glioblastoma with reduction of PAM expression in engrafted tumor significantly increasing the survival in mice. In summary, our data revealed PAM as a potential target for antiangiogenic therapy in glioblastoma.

Glioblastoma multiforme restructures the topological connectivity of cerebrovascular networks.

Glioblastoma multiforme alters healthy tissue vasculature by inducing angiogenesis and vascular remodeling. To fully comprehend the structural and functional properties of the resulting vascular network, it needs to be studied collectively by considering both geometric and topological properties. Utilizing Single Plane Illumination Microscopy (SPIM), the detailed capillary structure in entire healthy and tumor-bearing mouse brains could be resolved in three dimensions. At the scale of the smallest capillaries, the entire vascular systems of bulk U87- and GL261-glioblastoma xenografts, their respective cores, and healthy brain hemispheres were modeled as complex networks and quantified with fundamental topological measures. All individual vessel segments were further quantified geometrically and modular clusters were uncovered and characterized as meta-networks, facilitating an analysis of large-scale connectivity. An inclusive comparison of large tissue sections revealed that geometric properties of individual vessels were altered in glioblastoma in a relatively subtle way, with high intra- and inter-tumor heterogeneity, compared to the impact on the vessel connectivity. A network topology analysis revealed a clear decomposition of large modular structures and hierarchical network organization, while preserving most fundamental topological classifications, in both tumor models with distinct growth patterns. These results augment our understanding of cerebrovascular networks and offer a topological assessment of glioma-induced vascular remodeling. The findings may help understand the emergence of hypoxia and necrosis, and prove valuable for therapeutic interventions such as radiation or antiangiogenic therapy.

RhoA regulates translation of the Nogo-A decoy SPARC in white matter-invading glioblastomas.

Glioblastomas strongly invade the brain by infiltrating into the white matter along myelinated nerve fiber tracts even though the myelin protein Nogo-A prevents cell migration by activating inhibitory RhoA signaling. The mechanisms behind this long-known phenomenon remained elusive so far, precluding a targeted therapeutic intervention. This study demonstrates that the prevalent activation of AKT in gliomas increases the ER protein-folding capacity and enables tumor cells to utilize a side effect of RhoA activation: the perturbation of the IRE1α-mediated decay of SPARC mRNA. Once translation is initiated, glioblastoma cells rapidly secrete SPARC to block Nogo-A from inhibiting migration via RhoA. By advanced ultramicroscopy for studying single-cell invasion in whole, undissected mouse brains, we show that gliomas require SPARC for invading into white matter structures. SPARC depletion reduces tumor dissemination that significantly prolongs survival and improves response to cytostatic therapy. Our finding of a novel RhoA-IRE1 axis provides a druggable target for interfering with SPARC production and underscores its therapeutic value.

Genetic and Functional Characterization of ADAMTS13 Variants in a Patient Cohort with Upshaw-Schulman Syndrome Investigated in Germany.

Upshaw-Schulman syndrome (USS) is caused by severe ADAMTS13 (a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13) deficiency due to homozygous or compound heterozygous mutations in the ADAMTS13 gene. Previous studies suggest three possible disease mechanisms: (1) reduced secretion of ADAMTS13 variants, (2) impaired proteolytic activity, (3) defective biosynthesis due to nonsense-mediated decay. Expression studies have failed to establish a clear genotype/phenotype correlation that could explain the significant variability in the age of onset and patients' clinical courses. In this study, we investigated ADAMTS13 sequence variations in 30 USS patients and identified 31 disease-causing mutations; among them 10 novel variants. While none of the recombinant proteins exhibited significant retention in the endoplasmic reticulum, secretion and activity analysis revealed defective release for all but one missense mutant. The latter exhibited normal secretion but impaired activity due to inactivation of the catalytic domain. Truncated mutants showed secretion and residual activity even though the patients suffered from a severe phenotype. The expression systems which we used may not be appropriate here, as they do not assess nonsense-mediated decay causing degradation of mRNA. In some patients, phenotypic severity could be explained by the combined effects of two mutations. Genetic screening in combination with in vitro characterization of ADAMTS13 variants from both alleles is a valuable tool to predict the phenotypic severity of USS. When necessary, supplementary methods, such as kinetics under flow conditions and mRNA processing assays, can be included. Such data are helpful to identify patients who are at high risk for severe attacks and therefore might benefit from prophylactic treatment.

Single- and two-photon imaging of human micrometastases and disseminated tumour cells with conjugates of nanobodies and quantum dots.

Early detection of malignant tumours and, especially, micrometastases and disseminated tumour cells is still a challenge. In order to implement highly sensitive diagnostic tools we demonstrate the use of nanoprobes engineered from nanobodies (single-domain antibodies, sdAbs) and fluorescent quantum dots (QDs) for single- and two-photon detection and imaging of human micrometastases and disseminated tumour cells in ex vivo biological samples of breast and pancreatic metastatic tumour mouse models expressing human epidermal growth factor receptor 2 (HER2) or carcinoembryonic antigen (CEA). By staining thin (5-10 µm) paraffin and thick (50 µm) agarose tissue sections, we detected HER2- and CEA-positive human tumour cells infiltrating the surrounding tissues or metastasizing to different organs, including the brain, testis, lung, liver, and lymph nodes. Compared to conventional fluorescently labelled antibodies the sdAb-HER2-QD and sdAb-CEA-QD nanoprobes are superior in detecting micrometastases in tissue sections by lower photobleaching and higher brightness of fluorescence signals ensuring much better discrimination of positive signals versus background. Very high two-photon absorption cross-sections of QDs and small size of the nanoprobes ensure efficient imaging of thick tissue sections unattainable with conventional fluorescent probes. The nanobody-QD probes will help to improve early cancer diagnosis and prognosis of progression by assessing metastasis.

A PRDX1-p38α heterodimer amplifies MET-driven invasion of IDH-wildtype and IDH-mutant gliomas.

The Peroxiredoxin 1 (PRDX1) gene maps to chromosome arm 1p and is hemizygously deleted and epigenetically silenced in isocitrate dehydrogenase 1 or 2 (IDH)-mutant and 1p/19q-codeleted oligodendroglial tumors. In contrast, IDH-wildtype astrocytic gliomas including glioblastomas mostly lack epigenetic silencing and express PRDX1 protein. In our study, we investigated how PRDX1 contributes to the infiltrative growth of IDH-wildtype gliomas. Focusing on p38α-dependent pathways, we analyzed clinical data from 133 patients of the NOA-04 trial cohort to look for differences in the gene expression profiles of gliomas with wildtype or mutant IDH. Biochemical interaction studies as well as in vitro and ex vivo migration studies were used to establish a biological role of PRDX1 in maintaining pathway activity. Whole-brain high-resolution ultramicroscopy and survival analyses of pre-clinical mouse models for IDH-wildtype gliomas were then used for in vivo confirmation. Based on clinical data, we found that the absence of PRDX1 is associated with changes in the expression of MET/HGF signaling components. PRDX1 forms a heterodimer with p38α mitogen-activated protein kinase 14 (MAPK14), stabilizing phospho-p38α in glioma cells. This process amplifies hepatocyte growth factor (HGF)-mediated signaling and stimulates actin cytoskeleton dynamics that promote glioma cell migration. Whole-brain high-resolution ultramicroscopy confirms these findings, indicating that PRDX1 promotes glioma brain invasion in vivo. Finally, reduced expression of PRDX1 increased survival in mouse glioma models. Thus, our preclinical findings suggest that PRDX1 expression levels may serve as a molecular marker for patients who could benefit from targeted inhibition of MET/HGF signaling.

Correlated MRI and Ultramicroscopy (MR-UM) of Brain Tumors Reveals Vast Heterogeneity of Tumor Infiltration and Neoangiogenesis in Preclinical Models and Human Disease.

Diffuse tumor infiltration into the adjacent parenchyma is an effective dissemination mechanism of brain tumors. We have previously developed correlated high field magnetic resonance imaging and ultramicroscopy (MR-UM) to study neonangiogenesis in a glioma model. In the present study we used MR-UM to investigate tumor infiltration and neoangiogenesis in a translational approach. We compare infiltration and neoangiogenesis patterns in four brain tumor models and the human disease: whereas the U87MG glioma model resembles brain metastases with an encapsulated growth and extensive neoangiogenesis, S24 experimental gliomas mimic IDH1 wildtype glioblastomas, exhibiting infiltration into the adjacent parenchyma and along white matter tracts to the contralateral hemisphere. MR-UM resolves tumor infiltration and neoangiogenesis longitudinally based on the expression of fluorescent proteins, intravital dyes or endogenous contrasts. Our study demonstrates the huge morphological diversity of brain tumor models regarding their infiltrative and neoangiogenic capacities and further establishes MR-UM as a platform for translational neuroimaging.

Classical and Novel TSPO Ligands for the Mitochondrial TSPO Can Modulate Nuclear Gene Expression: Implications for Mitochondrial Retrograde Signaling.

It is known that knockdown of the mitochondrial 18 kDa translocator protein (TSPO) as well as TSPO ligands modulate various functions, including functions related to cancer. To study the ability of TSPO to regulate gene expression regarding such functions, we applied microarray analysis of gene expression to U118MG glioblastoma cells. Within 15 min, the classical TSPO ligand PK 11195 induced changes in expression of immediate early genes and transcription factors. These changes also included gene products that are part of the canonical pathway serving to modulate general gene expression. These changes are in accord with real-time, reverse transcriptase (RT) PCR. At the time points of 15, 30, 45, and 60 min, as well as 3 and 24 h of PK 11195 exposure, the functions associated with the changes in gene expression in these glioblastoma cells covered well known TSPO functions. These functions included cell viability, proliferation, differentiation, adhesion, migration, tumorigenesis, and angiogenesis. This was corroborated microscopically for cell migration, cell accumulation, adhesion, and neuronal differentiation. Changes in gene expression at 24 h of PK 11195 exposure were related to downregulation of tumorigenesis and upregulation of programmed cell death. In the vehicle treated as well as PK 11195 exposed cell cultures, our triple labeling showed intense TSPO labeling in the mitochondria but no TSPO signal in the cell nuclei. Thus, mitochondrial TSPO appears to be part of the mitochondria-to-nucleus signaling pathway for modulation of nuclear gene expression. The novel TSPO ligand 2-Cl-MGV-1 appeared to be very specific regarding modulation of gene expression of immediate early genes and transcription factors.

In vivo nanoparticle imaging of innate immune cells can serve as a marker of disease severity in a model of multiple sclerosis.

Innate immune cells play a key role in the pathogenesis of multiple sclerosis and experimental autoimmune encephalomyelitis (EAE). Current clinical imaging is restricted to visualizing secondary effects of inflammation, such as gliosis and blood-brain barrier disruption. Advanced molecular imaging, such as iron oxide nanoparticle imaging, can allow direct imaging of cellular and molecular activity, but the exact cell types that phagocytose nanoparticles in vivo and how phagocytic activity relates to disease severity is not well understood. In this study we used MRI to map inflammatory infiltrates using high-field MRI and fluorescently labeled cross-linked iron oxide nanoparticles for cell tracking. We confirmed nanoparticle uptake and MR detectability ex vivo. Using in vivo MRI, we identified extensive nanoparticle signal in the cerebellar white matter and circumscribed cortical gray matter lesions that developed during the disease course (4.6-fold increase of nanoparticle accumulation in EAE compared with healthy controls, P < 0.001). Nanoparticles showed good cellular specificity for innate immune cells in vivo, labeling activated microglia, infiltrating macrophages, and neutrophils, whereas there was only sparse uptake by adaptive immune cells. Importantly, nanoparticle signal correlated better with clinical disease than conventional gadolinium (Gd) imaging (r, 0.83 for nanoparticles vs. 0.71 for Gd-imaging, P < 0.001). We validated our approach using the Food and Drug Administration-approved iron oxide nanoparticle ferumoxytol. Our results show that noninvasive molecular imaging of innate immune responses can serve as an imaging biomarker of disease activity in autoimmune-mediated neuroinflammation with potential clinical applications in a wide range of inflammatory diseases.

Ultramicroscopy as a novel tool to unravel the tropism of AAV gene therapy vectors in the brain.

Recombinant adeno-associated viral (AAV) vectors have advanced to the vanguard of gene therapy. Numerous naturally occurring serotypes have been used to target cells in various tissues. There is a strong need for fast and dynamic methods which efficiently unravel viral tropism in whole organs. Ultramicroscopy (UM) is a novel fluorescence microscopy technique that images optically cleared undissected specimens, achieving good resolutions at high penetration depths while being non-destructive. UM was applied to obtain high-resolution 3D analysis of AAV transduction in adult mouse brains, especially in the hippocampus, a region of interest for Alzheimer's disease therapy. We separately or simultaneously compared transduction efficacies for commonly used serotypes (AAV9 and AAVrh10) using fluorescent reporter expression. We provide a detailed comparative and quantitative analysis of the transduction profiles. UM allowed a rapid analysis of marker fluorescence expression in neurons with intact projections deep inside the brain, in defined anatomical structures. Major hippocampal neuronal transduction was observed with both vectors, with slightly better efficacy for AAV9 in UM. Glial response and synaptic marker expression did not change post transduction.We propose UM as a novel valuable complementary tool to efficiently and simultaneously unravel tropism of different viruses in a single non-dissected adult rodent brain.

In vivo detection of small tumour lesions by multi-pinhole SPECT applying a (99m)Tc-labelled nanobody targeting the Epidermal Growth Factor Receptor.

The detection of tumours in an early phase of tumour development in combination with the knowledge of expression of tumour markers such as epidermal growth factor receptor (EGFR) is an important prerequisite for clinical decisions. In this study we applied the anti-EGFR nanobody (99m)Tc-D10 for visualizing small tumour lesions with volumes below 100 mm(3) by targeting EGFR in orthotopic human mammary MDA-MB-468 and MDA-MB-231 and subcutaneous human epidermoid A431 carcinoma mouse models. Use of nanobody (99m)Tc-D10 of a size as small as 15.5 kDa enables detection of tumours by single photon emission computed tomography (SPECT) imaging already 45 min post intravenous administration with high tumour uptake (>3% ID/g) in small MDA-MB-468 and A431 tumours, with tumour volumes of 52.5 mm(3) ± 21.2 and 26.6 mm(3) ± 16.7, respectively. Fast blood clearance with a serum half-life of 4.9 min resulted in high in vivo contrast and ex vivo tumour to blood and tissue ratios. In contrast, no accumulation of (99m)Tc-D10 in MDA-MB-231 tumours characterized by a very low expression of EGFR was observed. Here we present specific and high contrast in vivo visualization of small human tumours overexpressing EGFR by preclinical multi-pinhole SPECT shortly after administration of anti-EGFR nanobody (99m)Tc-D10.

Correlated magnetic resonance imaging and ultramicroscopy (MR-UM) is a tool kit to assess the dynamics of glioma angiogenesis.

Neoangiogenesis is a pivotal therapeutic target in glioblastoma. Tumor monitoring requires imaging methods to assess treatment effects and disease progression. Until now mapping of the tumor vasculature has been difficult. We have developed a combined magnetic resonance and optical toolkit to study neoangiogenesis in glioma models. We use in vivo magnetic resonance imaging (MRI) and correlative ultramicroscopy (UM) of ex vivo cleared whole brains to track neovascularization. T2* imaging allows the identification of single vessels in glioma development and the quantification of neovessels over time. Pharmacological VEGF inhibition leads to partial vascular normalization with decreased vessel caliber, density, and permeability. To further resolve the tumor microvasculature, we performed correlated UM of fluorescently labeled microvessels in cleared brains. UM resolved typical features of neoangiogenesis and tumor cell invasion with a spatial resolution of ~5 µm. MR-UM can be used as a platform for three-dimensional mapping and high-resolution quantification of tumor angiogenesis.