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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".

Antibody-based therapies hold promise for a safe and efficient treatment of cancer. The identification of target tumor cells through a specific antigen enriched on their surface and the subsequent delivery of the therapeutic agent only to those cells requires, besides the efficacy of the therapeutic agent itself, the identification of an antigen enriched on the surface of tumor cells, the generation of high affinity antibodies against that antigen. We have generated single-domain antibodies (nanobodies) against the voltage-gated potassium channel Kv10.1, which outside of the brain is detectable almost exclusively in tumor cells. The nanobody with highest affinity was fused to an improved form of the tumor necrosis factor-related apoptosis inducing ligand TRAIL, to target this cytokine to the surface of tumor cells. The resulting construct, VHH-D9-scTRAIL, shows rapid and strong apoptosis induction in different tumor models in cell culture. The construct combines two sources of specificity, the expression of the antigen restricted to tumor cells and the tumor selectivity of TRAIL. Such specificity combined with the high affinity obtained through nanobodies make the novel agent a promising concept for cancer therapy., (Copyright © 2020 Hartung, Krüwel, Shi, Pfizenmaier, Kontermann, Chames, Alves and Pardo.)

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.

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.

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., Competing Interests: The authors declare no conflict of interest.

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.

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.