Glioblastoma PET/MRI: kinetic investigation of [18F]rhPSMA-7.3, [18F]FET and [18F]fluciclovine in an orthotopic mouse model of cancer.

PURPOSE: Glioblastoma multiforme (GBM) is the most common glioma and standard therapies can only slightly prolong the survival. Neo-vascularization is a potential target to image tumor microenvironment, as it defines its brain invasion. We investigate [18F]rhPSMA-7.3 with PET/MRI for quantitative imaging of neo-vascularization in GBM bearing mice and human tumor tissue and compare it to [18F]FET and [18F]fluciclovine using PET pharmacokinetic modeling (PKM). METHODS: [18F]rhPSMA-7.3, [18F]FET, and [18F]fluciclovine were i.v. injected with 10.5 ± 3.1 MBq, 8.0 ± 2.2 MBq, 11.5 ± 1.9 MBq (n = 28, GL261-luc2) and up to 90 min PET/MR imaged 21/28 days after surgery. Regions of interest were delineated on T2-weighted MRI for (i) tumor, (ii) brain, and (iii) the inferior vena cava. Time-activity curves were expressed as SUV mean, SUVR and PKM performed using 1-/2-tissue-compartment models (1TCM, 2TCM), Patlak and Logan analysis (LA). Immunofluorescent staining (IFS), western blotting, and autoradiography of tumor tissue were performed for result validation. RESULTS: [18F]rhPSMA-7.3 showed a tumor uptake with a tumor-to-background-ratio (TBR) = 2.1-2.5, in 15-60 min. PKM (2TCM) confirmed higher K1 (0.34/0.08, p = 0.0012) and volume of distribution VT (0.24/0.1, p = 0.0017) in the tumor region compared to the brain. Linearity in LA and similar k3 = 0.6 and k4 = 0.47 (2TCM, tumor, p = ns) indicated reversible binding. K1, an indicator for vascularization, increased (0.1/0.34, 21 to 28 days, p < 0.005). IFS confirmed co-expression of PSMA and tumor vascularization. [18F]fluciclovine showed higher TBR (2.5/1.8, p < 0.001, 60 min) and VS (1.3/0.7, p < 0.05, tumor) compared to [18F]FET and LA indicated reversible binding. VT increased (p < 0.001, tumor, 21 to 28 days) for [18F]FET (0.5-1.4) and [18F]fluciclovine (0.84-1.5). CONCLUSION: [18F]rhPSMA-7.3 showed to be a potential candidate to investigate the tumor microenvironment of GBM. Following PKM, this uptake was associated with tumor vascularization. In contrast to what is known from PSMA-PET in prostate cancer, reversible binding was found for [18F]rhPSMA-7.3 in GBM, contradicting cellular trapping. Finally, [18F]fluciclovine was superior to [18F]FET rendering it more suitable for PET imaging of GBM.

Positron emission tomography and single photon emission computed tomography imaging of tertiary lymphoid structures during the development of lupus nephritis.

Lymphoid neogenesis occurs in tissues targeted by chronic inflammatory processes, such as infection and autoimmunity. In systemic lupus erythematosus (SLE), such structures develop within the kidneys of lupus-prone mice ((NZBXNZW)F1) and are observed in kidney biopsies taken from SLE patients with lupus nephritis (LN). The purpose of this prospective longitudinal animal study was to detect early kidney changes and tertiary lymphoid structures (TLS) using in vivo imaging. Positron emission tomography (PET) by tail vein injection of 18-F-fluoro-2-deoxy-D-glucose (18F-FDG)(PET/FDG) combined with computed tomography (CT) for anatomical localization and single photon emission computed tomography (SPECT) by intraperitoneal injection of 99mTC labeled Albumin Nanocoll (99mTC-Nanocoll) were performed on different disease stages of NZB/W mice (n = 40) and on aged matched control mice (BALB/c) (n = 20). By using one-way ANOVA analyses, we compared two different compartmental models for the quantitative measure of 18F-FDG uptake within the kidneys. Using a new five-compartment model, we observed that glomerular filtration of 18FFDG in lupus-prone mice decreased significantly by disease progression measured by anti-dsDNA Ab production and before onset of proteinuria. We could not visualize TLS within the kidneys, but we were able to visualize pancreatic TLS using 99mTC Nanocoll SPECT. Based on our findings, we conclude that the five-compartment model can be used to measure changes of FDG uptake within the kidney. However, new optimal PET/SPECT tracer administration sites together with more specific tracers in combination with magnetic resonance imaging (MRI) may make it possible to detect formation of TLS and LN before clinical manifestations.

Machine learning derived input-function in a dynamic 18F-FDG PET study of mice.

Tracer kinetic modelling, based on dynamic 18F-fluorodeoxyglucose (FDG) positron emission tomography (PET) is used to quantify glucose metabolism in humans and animals. Knowledge of the arterial input-function (AIF) is required for such measurements. Our aim was to explore two non-invasive machine learning-based models, for AIF prediction in a small-animal dynamic FDG PET study. 7 tissue regions were delineated in images from 68 FDG PET/computed tomography mouse scans. Two machine learning-based models were trained for AIF prediction, based on Gaussian processes (GP) and a long short-term memory (LSTM) recurrent neural network, respectively. Because blood data were unavailable, a reference AIF was formed by fitting an established AIF model to vena cava and left ventricle image data. The predicted and reference AIFs were compared by the area under curve (AUC) and root mean square error (RMSE). Net-influx rate constants, K i , were calculated with a two-tissue compartment model, using both predicted and reference AIFs for three tissue regions in each mouse scan, and compared by means of error, ratio, correlation coefficient, P value and Bland-Altman analysis. The impact of different tissue regions on AIF prediction was evaluated by training a GP and an LSTM model on subsets of tissue regions, and calculating the RMSE between the reference and the predicted AIF curve. Both models generated AIFs with AUCs similar to reference. The LSTM models resulted in lower AIF RMSE, compared to GP. K i from both models agreed well with reference values, with no significant differences. Myocardium was highlighted as important for AIF prediction, but AIFs with similar RMSE were obtained also without myocardium in the input data. Machine learning can be used for accurate and non-invasive prediction of an image-derived reference AIF in FDG studies of mice. We recommend the LSTM approach, as this model predicts AIFs with lower errors, compared to GP.

Evaluation by metabolic profiling and in vitro autoradiography of two promising GnRH-receptor ligands for brain SPECT imaging.

The increased expression of gonadotropin releasing hormone receptor (GnRH-R) in brain has been strongly linked to Alzheimer disease. Therefore, the development of radiolabeled imaging agents for GnRH-R is relevant for early diagnosis of Alzheimer disease. We have recently disclosed the discovery of two promising compounds displaying nanomolar-range affinity for the GnRH-R. In the present study, a preclinical evaluation of the compound properties was performed to evaluate their potential as single photon emission computed tomography (SPECT) radiotracers for imaging the GnRH-receptor. The compounds were assessed in vitro by performing serum stability analysis by human and rat serum, metabolic profiling by human liver microsomes, and exploratory rat brain autoradiography. The investigated compounds displayed satisfactory stability against human, rat serum, and liver microsomal metabolism, which favors their potential as SPECT-imaging agents. Additionally, we identified and quantified the formation rate of the metabolites by fragmentation of up to five mass spectrometric stages. The GnRH-R rat brain specificity of these compounds was tested in competition with a known ligand for the receptor and the in vitro autoradiography confirmed that compounds 3 and 4 binds to rat GnRH-R in different rat brain regions.

First in vivo evaluation of a potential SPECT brain radiotracer for the gonadotropin releasing hormone receptor.

OBJECTIVES: In vivo evaluations of a gonadotropin releasing hormone-receptor single photon emission computed tomography radiotracer for non-invasive detection of gonadotropin releasing homone-receptors in brain. RESULTS: We have used a simple, robust and high-yielding procedure to radiolabel an alpha-halogenated bioactive compound with high radiochemical yield. Literature findings showed similar alpha-halogenated compounds suitable for in vivo evaluations. The compound was found to possess nano molar affinity for the gonadotropin releasing hormone-receptor in a competition dependent inhibition study. Furthermore, liquid chromatography-mass spectrometry analysis in saline, human and rat serum resulted in 46%, 52% and 44% stability after incubation for 1 h respectively. In addition, rat brain single photon emission computed tomography and biodistribution studies gave further insight into the nature of the compound as a radiotracer.

The interplay of matrix metalloproteinase-8, transforming growth factor-ß1 and vascular endothelial growth factor-C cooperatively contributes to the aggressiveness of oral tongue squamous cell carcinoma.

BACKGROUND: Matrix metalloproteinase-8 (MMP-8) has oncosuppressive properties in various cancers. We attempted to assess MMP-8 function in oral tongue squamous cell carcinoma (OTSCC). METHODS: MMP-8 overexpressing OTSCC cells were used to study the effect of MMP-8 on proliferation, apoptosis, migration, invasion and gene and protein expression. Moreover, MMP-8 functions were assessed in the orthotopic mouse tongue cancer model and by immunohistochemistry in patient samples. RESULTS: MMP-8 reduced the invasion and migration of OTSCC cells and decreased the expression of MMP-1, cathepsin-K and vascular endothelial growth factor-C (VEGF-C). VEGF-C was induced by transforming growth factor-ß1 (TGF-ß1) in control cells, but not in MMP-8 overexpressing cells. In human OTSCC samples, low MMP-8 in combination with high VEGF-C was an independent predictor of poor cancer-specific survival. TGF-ß1 treatment also restored the migration of MMP-8 overexpressing cells to the level of control cells. In mouse tongue cancer, MMP-8 did not inhibit metastasis, possibly because it was eliminated in the peripheral carcinoma cells. CONCLUSIONS: The suppressive effects of MMP-8 in OTSCC may be mediated through interference of TGF-ß1 and VEGF-C function and altered proteinase expression. Together, low MMP-8 and high VEGF-C expression have strong independent prognostic value in OTSCC.

The role of CD44 in fetal and adult hematopoietic stem cell regulation.

Throughout development, hematopoietic stem cells migrate to specific microenvironments, where their fate is, in part, extrinsically controlled. CD44 standard as a member of the cell adhesion molecule family is extensively expressed within adult bone marrow and has been previously reported to play important roles in adult hematopoietic regulation via CD44 standard-ligand interactions. In this manuscript, CD44 expression and function are further assessed and characterized on both fetal and adult hematopoietic stem cells. Using a CD44(-/-) mouse model, conserved functional roles of CD44 are revealed throughout development. CD44 is critical in the maintenance of hematopoietic stem and progenitor pools, as well as in hematopoietic stem cell migration. CD44 expression on hematopoietic stem cells as well as other hematopoietic cells within the bone marrow microenvironment is important in the homing and lodgment of adult hematopoietic stem cells isolated from the bone/bone marrow interface. CD44 is also involved in fetal hematopoietic stem cell migration out of the liver, via a process involving stromal cell-derived factor-1α. The absence of CD44 in neonatal bone marrow has no impact on the size of the long-term reconstituting hematopoietic stem cell pool, but results in an enhanced long-term engraftment potential of hematopoietic stem cells.

Brief Report: Factors Released by Megakaryocytes Thrombin Cleave Osteopontin to Negatively Regulate Hematopoietic Stem Cells.

Factor V (FV) and factor X (FX) activate and complex to form prothrombinase which subsequently cleaves prothrombin (PT), converting it to active thrombin. Thrombin cleaved osteopontin (tcOPN) contains a cryptic binding site for α4 ß1 and α9 ß1 integrins. We have previously shown that hematopoietic stem cells (HSC) bind to tcOPN via this site resulting in a decrease in their proliferation and differentiation. Therefore, tcOPN and the factors required for its generation are important components of the HSC niche. Herein we show mature megakaryocytes (MM, ≥8N) contain FV, FX, and PT mRNA and protein. Furthermore, we show 8N, 16N, 32N, and 64N MM all release the required factors to enable thrombin cleavage of OPN. Importantly, mice devoid of the myeloproliferative leukemia protein (Mpl), c-Mpl(-/-) mice, contain only approximately 10% of normal megakaryocyte numbers, showed significantly reduced FX and tcOPN protein levels in endosteal bone marrow (BM). In addition, WT hematopoietic progenitors and HSC showed reduced homing to the BM of c-Mpl(-/-) mice. This is the first report identifying MM as a key cellular component in the production of tcOPN in situ, allowing the BM microenvironment to self regulate HSC biology via tcOPN.

Isolation of murine bone marrow scavenging sinusoidal endothelial cells.

The bone marrow (BM) is permeated with sinusoidal vessels lined with sinusoidal endothelial cells (SEC), which are crucial for BM physiology and hemopoietic stem cell (HSC) renewal. However, little is known about the characteristics or functional capacity of bone marrow sinusoidal endothelial cells (BMSEC). One significant barrier to the study of BMSEC is the lack of specific cell surface markers that can be used to isolate these cells. Nevertheless, BMSEC possess one exceptional property, namely, the ability to scavenge large amounts of soluble waste molecules such as advanced glycation end-products (AGE) and we have utilized this to label BMSEC for cell sorting and isolation. We describe the means to produce and fluorescently label AGE, its use as a functional in vivo marker of BMSEC and the isolation of these cells from murine BM using fluorescent activated cell separation (FACS).

Squeezing red blood cells on an optical waveguide to monitor cell deformability during blood storage.

Red blood cells squeeze through micro-capillaries as part of blood circulation in the body. The deformability of red blood cells is thus critical for blood circulation. In this work, we report a method to optically squeeze red blood cells using the evanescent field present on top of a planar waveguide chip. The optical forces from a narrow waveguide are used to squeeze red blood cells to a size comparable to the waveguide width. Optical forces and pressure distributions on the cells are numerically computed to explain the squeezing process. The proposed technique is used to quantify the loss of blood deformability that occurs during blood storage lesion. Squeezing red blood cells using waveguides is a sensitive technique and works simultaneously on several cells, making the method suitable for monitoring stored blood.

Analyzing hematopoietic stem cell homing, lodgment, and engraftment to better understand the bone marrow niche.

The existence of a bone marrow (BM) niche--the location in which hematopoietic stem cells (HSCs) reside--was proposed more than 30 years ago. Recent data suggest that the interaction of HSCs with cellular and extracellular components within the BM is critical for HSC regulation. The tracking of immunofluorescently labeled, prospectively isolated HSCs to and within the BM cavity allows the assessment of the regulatory processes involved in (1) homing, which involves transendothelial migration into the BM; (2) lodgment, including transmarrow migration through the extravascular space; and (3) BM reconstitution. Together, such analyses provide a better understanding of the cellular and extracellular components involved in the regulation of HSC quiescence and differentiation. Homing and lodgment of transplanted HSCs, the first critical steps in engraftment, involve multiple interactions between HSCs and the BM microenvironment. Herein, we describe a refined method of analyzing homing efficiency and spatial distribution of HSCs harvested from endosteal and/or central BM regions; we also review alternate methods. Using these techniques, microenvironment modifications within the recipient or surface protein-expression modifications on donor HSCs in animal models provide insights into components influencing the homing, lodgment, and engraftment processes.

Understanding the role of the microenvironment during definitive hemopoietic development.

Hemopoietic stem cells (HSCs) are sustained in a specific microenvironment known as the stem cell niche. Recent studies in adult bone marrow have identified osteoblasts and endothelial cells as two important supportive cell types within the niche and demonstrated that interactions between HSCs and cellular and extracellular components within the endosteal and perivascular regions are critical for HSC regulation. However, the understanding of the role of the microenvironment in definitive HSC establishment, expansion, and maintenance during embryonic development is extremely limited. This review focuses on what is known about the components of each HSC microenvironment at various developmental stages and their known functional roles.

Hepatic disposal of advanced glycation end products during maturation and aging.

UNLABELLED: Aging is characterized by progressive loss of metabolic and biochemical functions and accumulation of metabolic by-products, including advanced glycation end products (AGEs), which are observed in several pathological conditions. A number of waste macromolecules, including AGEs are taken up from the circulation by endocytosis mainly into liver sinusoidal endothelial cells (LSECs) and Kupffer cells (KCs). However, AGEs still accumulate in different tissues with aging, despite the presence of this clearance mechanism. The aim of the present study was to determine whether the efficiency of LSECs and KCs for disposal of AGEs changes through aging. RESULTS: After intravenous administration of (14)C-AGE-albumin in pre-pubertal, young adult, middle aged and old mice, more than 90% of total recovered (14)C-AGE was liver associated, irrespective of age. LSECs and KCs represented the main site of uptake. A fraction of the (14)C-AGE degradation products ((14)C-AGE-DPs) was stored for months in the lysosomes of these cells after uptake. The overall rate of elimination of (14)C-AGE-DPs from the liver was markedly faster in pre-pubertal than in all post-pubertal age groups. The ability to eliminate (14)C-AGE-DPs decreased to similar extents after puberty in LSECs and KCs. A rapid early removal phase was characteristic for all age groups except the old group, where this phase was absent. CONCLUSIONS: Removal of AGE-DPs from the liver scavenger cells is a very slow process that changes with age. The ability of these cells to dispose of AGEs declines after puberty. Decreased AGE removal efficiency early in life may lead to AGE accumulation.

Effects of oxidized low-density lipoproteins on the hepatic microvasculature.

Oxidized low-density lipoproteins (oxLDLs) are involved in proinflammatory and cytotoxic events in different microcirculatory systems. The liver is an important scavenger organ for circulating oxLDLs. However, the interaction of oxLDL with the hepatic microcirculation has been poorly investigated. The present study was conducted to examine the effects of differently modified oxLDLs on the hepatic microvasculature. C57Bl/6J mice were injected intravenously with low-density lipoprotein (LDL), or LDL oxidized for 3 h (oxLDL(3)) or 24 h (oxLDL(24)), at doses resembling oxLDL plasma levels in cardiovascular disease patients. Radioiodinated ligands were used to measure blood decay and organ distribution, and nonlabeled ligands to evaluate microcirculatory responses, examined by in vivo microscopy 30-60 min after ligand injection, immunohistochemistry, and scanning and transmission electron microscopy. Mildly oxLDL (oxLDL(3)) was cleared from blood at a markedly slower rate than heavily oxLDL (oxLDL(24)), but significantly faster than LDL (P < 0.01). Injected oxLDLs distributed to liver. OxLDL effects were most pronounced in central areas of the liver lobules where oxLDL(3) elicited a significant (P < 0.05) reduction in perfused sinusoids, and both oxLDL(3) and oxLDL(24) significantly increased the numbers of swollen endothelial cells and adherent leukocytes compared with LDL (P < 0.05). OxLDL-treated livers also exhibited increased intercellular adhesion molecule (ICAM)-1 centrilobular staining. Electron microscopy showed a 30% increased thickness of the liver sinusoidal endothelium in the oxLDL(3) group (P < 0.05) and a reduced sinusoidal fenestration in centrilobular areas with increased oxidation of LDL (P for linear trend <0.05). In conclusion, OxLDL induced several acute changes in the liver microvasculature, which may lead to sinusoidal endothelial dysfunction.

Role of liver sinusoidal endothelial cells and stabilins in elimination of oxidized low-density lipoproteins.

Atherogenesis is associated with elevated levels of low-density lipoprotein (LDL) and its oxidized form (oxLDL) in the blood. The liver is an important scavenger organ for circulating oxLDLs. The present study aimed to examine endocytosis of mildly oxLDL (the major circulating form of oxLDLs) in liver sinusoidal endothelial cells (LSECs) and the involvement of the scavenger receptors stabilin-1 and stabilin-2 in this process. Freshly isolated LSECs, Kupffer cells (KCs), and stabilin-1- and stabilin-2-transfected human embryonic kidney cells were incubated with fluorescently labeled or radiolabeled oxLDLs [oxidized for 3 h (oxLDL(3)), 6 h, or 24 h (oxLDL(24))] to measure endocytosis. The intracellular localization of oxLDLs and stabilins in LSECs was examined by immunofluorescence and immunogold electron microscopy. Whereas oxLDL(24) was endocytosed both by LSECs and KCs, oxLDL(3) (mildly oxLDL) was taken up by LSECs only. The LSEC uptake of oxLDLs was significantly inhibited by the scavenger receptor ligand formaldehyde-treated serum albumin. Uptake of all modified LDLs was high in stabilin-1-transfected cells, whereas stabilin-2-transfected cells preferentially took up oxLDL(24), suggesting that stabilin-1 is a more important receptor for mildly oxLDLs than stabilin-2. Double immunogold labeling experiments in LSECs indicated interactions of stabilin-1 and stabilin-2 with oxLDL(3) on the cell surface, in coated pits, and endocytic vesicles. LSECs but not KCs endocytosed mildly oxLDL. Both stabilin-1 and stabilin-2 were involved in the LSEC endocytosis of oxLDLs, but experiments with stabilin-transfected cells pointed to stabilin-1 as the most important receptor for mildly oxLDL.

Age-related changes in scavenger receptor-mediated endocytosis in rat liver sinusoidal endothelial cells.

Liver sinusoidal endothelial cells (LSECs) play an essential role in systemic waste clearance by effective endocytosis of blood-borne waste macromolecules. We aimed to study LSECs' scavenger function during aging, and whether age-related morphological changes (eg, defenestration) affect this function, in F344/BN F1 rats. Endocytosis of the scavenger receptor ligand formaldehyde-treated serum albumin was significantly reduced in LSECs from old rats. Ligand degradation, LSEC protein expression of the major scavenger receptors for formaldehyde-treated serum albumin endocytosis, stabilin-1 and stabilin-2, and their staining patterns along liver sinusoids, was similar at young and old age, suggesting that other parts of the endocytic machinery are affected by aging. Formaldehyde-treated serum albumin uptake per cell, and cell porosity evaluated by electron microscopy, was not correlated, indicating that LSEC defenestration is not linked to impaired endocytosis. We report a significantly reduced LSEC endocytic capacity at old age, which may be especially important in situations with increased circulatory waste loads.