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The tumor microenvironment is highly heterogeneous. For gliomas, the tumor-associated inflammatory response is pivotal to support growth and invasion. Factors of glioma growth, inflammation, and invasion, such as the translocator protein (TSPO) and matrix metalloproteinases (MMP), may serve as specific imaging biomarkers of the glioma microenvironment. In this study, noninvasive imaging by PET with [18F]DPA-714 (TSPO) and [18F]BR-351 (MMP) was used for the assessment of localization and quantification of the expression of TSPO and MMP. Imaging was performed in addition to established clinical imaging biomarker of active tumor volume ([18F]FET) in conjunction with MRI. We hypothesized that each imaging biomarker revealed distinct areas of the heterogeneous glioma tissue in a mouse model of human glioma. Tracers were found to be increased 1.4- to 1.7-fold, with [18F]FET showing the biggest volume as depicted by a thresholding-based, volumes of interest analysis. Tumor areas, which could not be detected by a single tracer and/or MRI parameter alone, were measured. Specific compartments of [18F]DPA-714 (14%) and [18F]BR-351 (11%) volumes along the tumor rim could be identified. [18F]DPA-714 (TSPO) and [18F]BR-351 (MMP) matched with histology. Glioma-associated microglia/macrophages (GAM) were identified as TSPO and MMP sources. Multitracer and multimodal molecular imaging approaches may allow us to gain important insights into glioma-associated inflammation (GAM, MMP). Moreover, this noninvasive technique enables characterization of the glioma microenvironment with respect to the disease-driving cellular compartments at the various disease stages. Cancer Res; 77(8); 1831–41. ©2017 AACR.

Gli36dEGFR cells were implanted. Thereafter, two imaging sequences were conducted: In sequence 1, T2w MRI and PET with [18F]DPA-714 was performed, while [18F]BR-351 PET and [18F]FET PET were conducted at day 13 and 14, respectively. In imaging sequence 2, T2w MRI and [18F]FET PET were conducted at day 12, followed by [18F]BR-351 and [18F]DPA-714 at day 13 and 14, respectively. After the last imaging experiment, mice were sacrificed and the brains immediately harvested for further analysis.

PET and CT images were co-registered using three CT-detectable, molecular sieve spheres (# 1-3) rinsed in radiotracer solution. After segmentation of spheres in the CT image (arrows), PET and CT images were superimposed by using the automated "Landmark tool" provided by the Vinci software. The "Magic tool" was applied for minor corrections of co-registrations. The resulting resliced PET image was saved for further processing. In a second step, MR and CT images were superimposed using the "Fusion- and Magic tool". For illustration MR is pseudocolored in red, while the CT was inverted in color (black). The CT contour was further superimposed to demonstrate co-registration accuracy. Selected bone structures were used as additional quality measures of image registration. The resliced CT image was saved with the new transformation matrix. In a last step, the before mentioned transformation of the CT (CTMR) was transferred to the PET, resulting in co-registration of PET/MR/CT. This workflow was repeated for every PET scan per mouse.

(A) T/B ratios, (B-D) tracer volumes, (E-J) areas of unique tracer uptake and overlapping regions. No significant effect on volumetric data was observed based on the order of imaging experiments.

Supplementary Figure Legends 1-5

(A) An elliptical VOI (yellow) was drawn in the striatum. (B) Another elliptical VOI (green) was placed over the affected hemisphere. (C) After application of a thresholding based on the control VOI. The same original VOIs (left and middle) were used for all animals for individual thresholding.

An exemplary subset of n=6 mice were randomly assessed for intra-rater reproducibility. (A-E) [18F]DPA-714 and [18F]FET images were on two consecutive days repeatedly co-registered. Volumetry was performed twice (coreg 2 and coreg 3) and compared to the data reported in the main text (coreg 1). Dotted lines represent the overall mean for all n=20 animals reported in the main text. No significant changes for all parameters, except for the "percentage of [18F]DPA-714 only" between coreg 1 and 3, was found over the three different time points of co-registration.

Abnormal hematopoiesis advances cardiovascular disease by generating excess inflammatory leukocytes that attack the arteries and the heart. The bone marrow niche regulates hematopoietic stem cell proliferation and hence the systemic leukocyte pool, but whether cardiovascular disease affects the hematopoietic organ’s microvasculature is unknown. Here we show that hypertension, atherosclerosis and myocardial infarction (MI) instigate endothelial dysfunction, leakage, vascular fibrosis and angiogenesis in the bone marrow, altogether leading to overproduction of inflammatory myeloid cells and systemic leukocytosis. Limiting angiogenesis with endothelial deletion of Vegfr2 (encoding vascular endothelial growth factor (VEGF) receptor 2) curbed emergency hematopoiesis after MI. We noted that bone marrow endothelial cells assumed inflammatory transcriptional phenotypes in all examined stages of cardiovascular disease. Endothelial deletion of Il6 or Vcan (encoding versican), genes shown to be highly expressed in mice with atherosclerosis or MI, reduced hematopoiesis and systemic myeloid cell numbers in these conditions. Our findings establish that cardiovascular disease remodels the vascular bone marrow niche, stimulating hematopoiesis and production of inflammatory leukocytes.

Introduction Dysregulated activity of matrix metalloproteinases (MMPs) drives a variety of pathophysiological conditions. Non-invasive imaging of MMP activity in vivo promises diagnostic and prognostic value. However, current targeting strategies by small molecules are typically limited with respect to the bioavailability of the labeled MMP binders in vivo. To this end, we here introduce and compare three chemical modifications of a recently developed barbiturate-based radiotracer with respect to bioavailability and potential to image MMP activity in vivo. Methods Barbiturate-based MMP inhibitors with an identical targeting unit but varying hydrophilicity were synthesized, labeled with technetium-99m, and evaluated in vitro and in vivo. Biodistribution and radiotracer elimination were determined in C57/BL6 mice by serial SPECT imaging. MMP activity was imaged in a MMP-positive subcutaneous xenograft model of human K1 papillary thyroid tumors. In vivo data were validated by scintillation counting, autoradiography, and MMP immunohistochemistry. Results We prepared three new 99mTc‐labeled MMP inhibitors, bearing either a glycine ([99mTc]MEA39), lysine ([99mTc]MEA61), or the ligand HYNIC with the ionic co-ligand TPPTS ([99mTc]MEA223) yielding gradually increasing hydrophilicity. [99mTc]MEA39 and [99mTc]MEA61 were rapidly eliminated via hepatobiliary pathways. In contrast, [99mTc]MEA223 showed delayed in vivo clearance and primary renal elimination. In a thyroid tumor xenograft model, only [99mTc]MEA223 exhibited a high tumor-to-blood ratio that could easily be delineated in SPECT images. Conclusion Introduction of HYNIC/TPPTS into the barbiturate lead structure ([99mTc]MEA223) results in delayed renal elimination and allows non-invasive MMP imaging with high signal-to-noise ratios in a papillary thyroid tumor xenograft model.

A sedentary lifestyle, chronic inflammation and leukocytosis increase atherosclerosis; however, it remains unclear whether regular physical activity influences leukocyte production. Here we show that voluntary running decreases hematopoietic activity in mice. Exercise protects mice and humans with atherosclerosis from chronic leukocytosis but does not compromise emergency hematopoiesis in mice. Mechanistically, exercise diminishes leptin production in adipose tissue, augmenting quiescence-promoting hematopoietic niche factors in leptin-receptor-positive stromal bone marrow cells. Induced deletion of the leptin receptor in Prrx1-creERT2; Leprfl/fl mice reveals that leptin’s effect on bone marrow niche cells regulates hematopoietic stem and progenitor cell (HSPC) proliferation and leukocyte production, as well as cardiovascular inflammation and outcomes. Whereas running wheel withdrawal quickly reverses leptin levels, the impact of exercise on leukocyte production and on the HSPC epigenome and transcriptome persists for several weeks. Together, these data show that physical activity alters HSPCs via modulation of their niche, reducing hematopoietic output of inflammatory leukocytes. The beneficial effects of exercise on cardiovascular disease are linked to decreased inflammation through crosstalk between adipose tissue and hematopoietic progenitor cells in the bone marrow.

Dysregulated activity of matrix metalloproteinases (MMPs) drives a variety of pathophysiological conditions. Non-invasive imaging of MMP activity in vivo promises diagnostic and prognostic value. However, current targeting strategies by small molecules are typically limited with respect to the bioavailability of the labeled MMP binders in vivo. To this end, we here introduce and compare three chemical modifications of a recently developed barbiturate-based radiotracer with respect to bioavailability and potential to image MMP activity in vivo.Barbiturate-based MMP inhibitors with an identical targeting unit but varying hydrophilicity were synthesized, labeled with technetium-99m, and evaluated in vitro and in vivo. Biodistribution and radiotracer elimination were determined in C57/BL6 mice by serial SPECT imaging. MMP activity was imaged in a MMP-positive subcutaneous xenograft model of human K1 papillary thyroid tumors. In vivo data were validated by scintillation counting, autoradiography, and MMP immunohistochemistry.We prepared three newIntroduction of HYNIC/TPPTS into the barbiturate lead structure ([

Rationale: Inflammatory stress induced by exposure to bacterial lipopolysaccharide causes hematopoietic stem cell expansion in the bone marrow niche, generating a cellular immune response. As an integral component of the hematopoietic stem cell niche, the bone marrow vasculature regulates the production and release of blood leukocytes, which protect the host against infection but also fuel inflammatory diseases. Objective: We aimed to develop imaging tools to explore vascular changes in the bone marrow niche during acute inflammation. Methods and Results: Using the TLR (Toll-like receptor) ligand lipopolysaccharide as a prototypical danger signal, we applied multiparametric, multimodality and multiscale imaging to characterize how the bone marrow vasculature adapts when hematopoiesis boosts leukocyte supply. In response to lipopolysaccharide, ex vivo flow cytometry and histology showed vascular changes to the bone marrow niche. Specifically, proliferating endothelial cells gave rise to new vasculature in the bone marrow during hypoxic conditions. We studied these vascular changes with complementary intravital microscopy and positron emission tomography/magnetic resonance imaging. Fluorescence and positron emission tomography integrin αVβ3 imaging signal increased during lipopolysaccharide-induced vascular remodeling. Vascular leakiness, quantified by albumin-based in vivo microscopy and magnetic resonance imaging, rose when neutrophils departed and hematopoietic stem and progenitor cells proliferated more vigorously. Conclusions: Introducing a tool set to image bone marrow either with cellular resolution or noninvasively within the entire skeleton, this work sheds light on angiogenic responses that accompany emergency hematopoiesis. Understanding and monitoring bone marrow vasculature may provide a key to unlock therapeutic targets regulating systemic inflammation.

Recruitment of leukocytes from the blood to sites of inflammation poses a promising target for new diagnostic and therapeutic approaches. We aimed to develop a novel method to non-invasively analyze molecular mechanisms of leukocyte migration in pre-clinical models of inflammation in vivo. Methods: We used the ER-HoxB8 system to transiently immortalize murine myeloid precursors from wildtype and CD18- as well as MRP14-deficient mice. A VLA4α-/- cell line was generated by CRISPR/Cas9-mediated gene editing. We analyzed the migration of wildtype and knockout leukocytes in vivo by optical and nuclear imaging in mice with irritant contact dermatitis, cutaneous granuloma, experimental arthritis and myocardial infarction. Results: Transient immortalization, gene editing and in vivo imaging can be combined to analyze migratory mechanisms of murine leukocytes, even for gene deletions resulting in lethal phenotypes in mice. We reliably confirmed known migratory defects of leukocytes deficient for the adhesion molecules CD18 or VLA4α. Also, using our new method we identified a new role of the most abundant calcium-binding proteins in phagocytes and major alarmins in many inflammatory diseases, MRP8 and MRP14, for transmigration in vivo. Conclusion: We provide a combinatorial approach to rapidly characterize molecular mechanisms of leukocyte recruitment in vivo, with the potential to aid in identification of diagnostic and therapeutic targets in inflammatory pathologies.

Contains fulltext : 193229.pdf (Publisher’s version ) (Open Access) Modulation of the inflammatory microenvironment after stroke opens a new avenue for the development of novel neurorestorative therapies in stroke. Understanding the spatio-temporal profile of (neuro-)inflammatory imaging biomarkers in detail thereby represents a crucial factor in the development and application of immunomodulatory therapies. The early integration of quantitative molecular imaging biomarkers in stroke drug development may provide key information about (i) early diagnosis and follow-up, (ii) spatio-temporal drug-target engagement (pharmacodynamic biomarker), (iii) differentiation of responders and non-responders in the patient cohort (inclusion/exclusion criteria; predictive biomarkers), and (iv) the mechanism of action. The use of targeted imaging biomarkers for may thus allow clinicians to decipher the profile of patient-specific inflammatory activity and the development of patient-tailored strategies for immunomodulatory and neuro-restorative therapies in stroke. Here, we highlight the recent developments in preclinical and clinical molecular imaging biomarkers of neuroinflammation (endothelial markers, microglia, MMPs, cell labeling, future developments) in stroke and outline how imaging biomarkers can be used in overcoming current translational roadblocks and attrition in order to advance new immunomodulatory compounds within the clinical pipeline.

The tumor microenvironment is highly heterogeneous. For gliomas, the tumor-associated inflammatory response is pivotal to support growth and invasion. Factors of glioma growth, inflammation, and invasion, such as the translocator protein (TSPO) and matrix metalloproteinases (MMP), may serve as specific imaging biomarkers of the glioma microenvironment. In this study, noninvasive imaging by PET with [18F]DPA-714 (TSPO) and [18F]BR-351 (MMP) was used for the assessment of localization and quantification of the expression of TSPO and MMP. Imaging was performed in addition to established clinical imaging biomarker of active tumor volume ([18F]FET) in conjunction with MRI. We hypothesized that each imaging biomarker revealed distinct areas of the heterogeneous glioma tissue in a mouse model of human glioma. Tracers were found to be increased 1.4- to 1.7-fold, with [18F]FET showing the biggest volume as depicted by a thresholding-based, volumes of interest analysis. Tumor areas, which could not be detected by a single tracer and/or MRI parameter alone, were measured. Specific compartments of [18F]DPA-714 (14%) and [18F]BR-351 (11%) volumes along the tumor rim could be identified. [18F]DPA-714 (TSPO) and [18F]BR-351 (MMP) matched with histology. Glioma-associated microglia/macrophages (GAM) were identified as TSPO and MMP sources. Multitracer and multimodal molecular imaging approaches may allow us to gain important insights into glioma-associated inflammation (GAM, MMP). Moreover, this noninvasive technique enables characterization of the glioma microenvironment with respect to the disease-driving cellular compartments at the various disease stages. Cancer Res; 77(8); 1831–41. ©2017 AACR.

Rationale: After a stroke, patients frequently experience altered systemic immunity resulting in peripheral immunosuppression and higher susceptibility to infections, which is at least partly attributed to lymphopenia. The mechanisms that profoundly change the systemic leukocyte repertoire after stroke are incompletely understood. Emerging evidence indicates that stroke alters hematopoietic output of the bone marrow. Objective: To explore the mechanisms that lead to defects of B lymphopoiesis after ischemic stroke. Methods and Results: We here report that ischemic stroke triggers brain-bone marrow communication via hormonal long-range signals that regulate hematopoietic B lineage decisions. Bone marrow fluorescence-activated cell sorter analyses and serial intravital microscopy indicate that transient middle cerebral artery occlusion in mice arrests B-cell development beginning at the pro-B-cell stage. This phenotype was not rescued in Myd88 −/− and TLR4 −/− mice with disrupted TLR (Toll-like receptor) signaling or after blockage of peripheral sympathetic nerves. Mechanistically, we identified stroke-induced glucocorticoid release as the main instigator of B lymphopoiesis defects. B-cell lineage-specific deletion of the GR (glucocorticoid receptor) in CD19-Cre loxP Nr3c1 mice attenuated lymphocytopenia after transient middle cerebral artery. In 20 patients with acute stroke, increased cortisol levels inversely correlated with blood lymphocyte numbers. Conclusions: Our data demonstrate that the hypothalamic-pituitary-adrenal axis mediates B lymphopoiesis defects after ischemic stroke.

Macrophages are ubiquitous cells that reside in all major tissues. Counter to long-held beliefs, we now know that resident macrophages in many organs are seeded during embryonic development and self-renew independently from blood monocytes. Under inflammatory conditions, those tissue macrophages are joined and sometimes replaced by recruited monocyte-derived macrophages. Macrophage function in steady state and disease depends on not only their developmental origin but also the tissue environment. Here, we discuss the ontogeny, function, and interplay of tissue-resident and monocyte-derived macrophages in various organs contributing to the development and progression of cardiovascular disease.

Background Recruitment and migration of phagocytes to the site of inflammation are key events in the onset of inflammation. Albeit crucial for pathogen elimination, tissue repair and restoration of tissue homeostasis, dysregulated phagocyte infiltration can also cause severe inflammatory disorders. Therefore, targeting and modulation of phagocyte infiltration represents a promising new approach to fight inflammatory disorders and diseases, such as rheumatoid arthritis. Additionally, non-invasive tracking of phagocyte migration to the site of inflammation could extend both scientific knowledge as well as the repertoire of diagnostic strategies in clinical use. Objectives The aim of this study was to establish a fluorescence reflectance imaging (FRI) based system to visualize and analyze migration properties of different cell populations in inflammatory disease models, like experimental arthritis, in vivo . Methods Immortalized murine myeloid progenitor ER-HoxB8 cells were differentiated to neutrophils or monocytes (1). Cells were labeled with the membrane-selective fluorescent dyes DIR (2) or DID, respectively. We analyzed viability and functionality of stained cells in vitro and investigated their ability to migrate to sites of inflammation in vivo in several mouse models - particularly in a collagen induced arthritis (CIA) mouse model - via fluorescence reflectance imaging (FRI). Using CRISPR-Cas9 technology we introduced targeted gene deletions for main adhesion molecules. Results Differentiated ER-HoxB8 cells could effectively be labeled with DIR or DID. Labeling of monocytes or neutrophils did not affect cellular viability or functionality in vitro . Subsequent in vivo imaging experiments allowed the visualization of migrated labeled phagocytes in different murine disease models, thereby cells could be detected at sites of inflammation with high sensitivity and specificity. In a CIA mouse model the amount of immigrated cells could even be associated closely to disease score and disease severity. Thus, the detection of immigration of labeled cells might also give hints about new inflammatory spots that are about to settle up before they can be detected macroscopically. Furthermore, differential cell labeling allowed direct quantitative comparison of differences in migration rates of wildtype and CD18 or CD49d knockout cells in vivo . Conclusions Specific and distinguishable labeling of diverse cell types allows in vivo tracking and subsequent quantification of migrated cells within the same animal. Targeted gene deletion allows analysis of molecular mechanisms relevant for leukocyte recruitment during different stages of arthritis. Correlation of the amount of immigrated cells to disease severity offers new opportunities to non-invasively detect and monitor inflammatory sites in vivo . References Wang GG, Calvo KR, Pasillas MP, Sykes DB, Hacker H, Kamps MP. Quantitative production of macrophages or neutrophils ex vivo using conditional Hoxb8. Nat Methods. 2006;3(4):287–93. Eisenblatter M, Ehrchen J, Varga G, Sunderkotter C, Heindel W, Roth J, et al. In vivo optical imaging of cellular inflammatory response in granuloma formation using fluorescence-labeled macrophages. J Nucl Med. 2009;50:1676–82. Disclosure of Interest None declared

The enzymes gelatinase A/matrix metalloproteinase-2 (MMP-2) and gelatinase B/MMP-9 are essential for induction of neuroinflammatory symptoms in experimental autoimmune encephalomyelitis (EAE), a mouse model of multiple sclerosis (MS); in the absence of these enzymes, the disease does not develop. We therefore investigated the cellular sources and relative contributions of MMP-2 and MMP-9 to disease at early stages of EAE induction. We demonstrated that MMP-9 from an immune cell source is required in EAE for initial infiltration of leukocytes into the central nervous system and that MMP-9 activity is a reliable marker of leukocyte penetration of the blood-brain barrier. We then developed a molecular imaging method to visualize MMP activity in the brain using fluorescent- and radioactive-labeled MMP inhibitors (MMPis) in EAE animals and used the radioactive MMP ligand for positron emission tomography (PET) imaging of MMP activity in patients with MS. In contrast to traditional T1-gadolinium contrast-enhanced MRI, MMPi-PET enabled tracking of MMP activity as a unique feature of early lesions and ongoing leukocyte infiltration. MMPi-PET therefore allows monitoring of the early steps of MS development and provides a sensitive, noninvasive means of following lesion formation and resolution in murine EAE and human MS.

Spinocerebellar ataxia type 3 (SCA3) is pathologically characterized by the formation of intranuclear aggregates which contain ataxin-3, the mutated protein in SCA3, in a specific subtype of neurons. It has been proposed that ataxin-3 is cleaved by proteolytic enzymes, in particular by calpains and caspases, eventually leading to the formation of aggregates. In our study, we examined the ability of calpains to cleave ataxin-3 in vitro and in vivo. We demonstrated in cell culture and mouse brain homogenates that cleavage of overexpressed ataxin-3 by calpains and in particular by calpain-2 occur and that polyglutamine expanded ataxin-3 is more sensitive to calpain degradation. Based on these results, we investigated the influence of calpains on the pathogenesis of SCA3 in vivo. For this purpose, we enhanced calpain activity in a SCA3 transgenic mouse model by knocking out the endogenous calpain inhibitor calpastatin. Double-mutant mice demonstrated an aggravated neurological phenotype with an increased number of nuclear aggregates and accelerated neurodegeneration in the cerebellum. This study confirms the critical importance of calcium-dependent calpain-type proteases in the pathogenesis of SCA3 and suggests that the manipulation of the ataxin-3 cleavage pathway and the regulation of intracellular calcium homeostasis may represent novel targets for therapeutic intervention in SCA3.

BACKGROUND Association between genetic variants located on human chromosome 8q24.21 with an increased risk for prostatic carcinoma has been well established. POU5F1P1, a processed pseudogene homologous to the pluripotency factor OCT4, is the only sequence with coding capacity in this region. The objective of this study was to investigate the POU5F1P1 expression in prostatic carcinoma and carcinoma surrounding prostatic tissue. METHODS RT-PCR and real-time PCR was used to measure the expression of POU5F1P1 relative to the expression of HPRT1 in cell lines, prostatic carcinoma and carcinoma surrounding prostatic tissue. The structure of the POU5F1P1 mRNA and the promoter sequence were elucidated by 5′-RACE experiments. The POU5F1P1 protein was shown with immunohistochemistry on prostate tissue. RESULTS POU5F1P1 was found to be the only member of the POU5F1 family to be expressed in prostate with over-expression in prostatic carcinoma compared to surrounding prostatic tissue probably because of an increased density of expressing cells. The POU5F1P1 expression is driven by a variety of promoter structures scattered over a genomic region of 860 kB. CONCLUSIONS The over-expression of POU5F1P1 in prostatic carcinoma in addition to its genomic location and the putative function of its gene product render POU5F1P1 a good candidate to harbour functional genetic variants which modulate prostatic cancer susceptibility. Prostate 70: 666–674, 2010. © 2009 Wiley-Liss, Inc.