Visualizing Cancer-Originating Acetate Uptake Through MCT1 in Reactive Astrocytes in the Glioblastoma Tumor Microenvironment.

BACKGROUND: Reactive astrogliosis is a hallmark of various brain pathologies, including neurodegenerative diseases and glioblastomas. However, the specific intermediate metabolites contributing to reactive astrogliosis remain unknown. This study investigated how glioblastomas induce reactive astrogliosis in the neighboring microenvironment and explores 11C-acetate PET as an imaging technique for detecting reactive astrogliosis. METHODS: Through in vitro, mouse models, and human tissue experiments, we examined the association between elevated 11C-acetate uptake and reactive astrogliosis in gliomas. We explored acetate from glioblastoma cells, which triggers reactive astrogliosis in neighboring astrocytes by upregulating MAO-B and MCT1 expression. We evaluated the presence of cancer stem cells in the reactive astrogliosis region of glioblastomas and assessed the correlation between the volume of 11C-acetate uptake beyond MRI and prognosis. RESULTS: Elevated 11C-acetate uptake is associated with reactive astrogliosis and astrocytic MCT1 in the periphery of glioblastomas in human tissues and mouse models. Glioblastoma cells exhibit increased acetate production as a result of glucose metabolism, with subsequent secretion of acetate. Acetate derived from glioblastoma cells induces reactive astrogliosis in neighboring astrocytes by increasing the expression of MAO-B and MCT1. We found cancer stem cells within the reactive astrogliosis at the tumor periphery. Consequently, a larger volume of 11C-acetate uptake beyond contrast-enhanced MRI was associated with worse prognosis. CONCLUSION: Our results highlight the role of acetate derived from glioblastoma cells in inducing reactive astrogliosis and underscore the potential value of 11C-acetate PET as an imaging technique for detecting reactive astrogliosis, offering important implications for the diagnosis and treatment of glioblastomas.

Astrocytic scar restricting glioblastoma via glutamate-MAO-B activity in glioblastoma-microglia assembloid.

BACKGROUND: Glial scar formation is a reactive glial response confining injured regions in a central nervous system. However, it remains challenging to identify key factors formulating glial scar in response to glioblastoma (GBM) due to complex glia-GBM crosstalk. METHODS: Here, we constructed an astrocytic scar enclosing GBM in a human assembloid and a mouse xenograft model. GBM spheroids were preformed and then co-cultured with microglia and astrocytes in 3D Matrigel. For the xenograft model, U87-MG cells were subcutaneously injected to the Balb/C nude female mice. RESULTS: Additional glutamate was released from GBM-microglia assembloid by 3.2-folds compared to GBM alone. The glutamate upregulated astrocytic monoamine oxidase-B (MAO-B) activity and chondroitin sulfate proteoglycans (CSPGs) deposition, forming the astrocytic scar and restricting GBM growth. Attenuating scar formation by the glutamate-MAO-B inhibition increased drug penetration into GBM assembloid, while reducing GBM confinement. CONCLUSIONS: Taken together, our study suggests that astrocytic scar could be a critical modulator in GBM therapeutics.

Visualizing reactive astrocyte-neuron interaction in Alzheimer's disease using 11C-acetate and 18F-FDG.

Reactive astrogliosis is a hallmark of Alzheimer's disease (AD). However, a clinically validated neuroimaging probe to visualize the reactive astrogliosis is yet to be discovered. Here, we show that PET imaging with 11C-acetate and 18F-fluorodeoxyglucose (18F-FDG) functionally visualizes the reactive astrocyte-mediated neuronal hypometabolism in the brains with neuroinflammation and AD. To investigate the alterations of acetate and glucose metabolism in the diseased brains and their impact on the AD pathology, we adopted multifaceted approaches including microPET imaging, autoradiography, immunohistochemistry, metabolomics, and electrophysiology. Two AD rodent models, APP/PS1 and 5xFAD transgenic mice, one adenovirus-induced rat model of reactive astrogliosis, and post-mortem human brain tissues were used in this study. We further curated a proof-of-concept human study that included 11C-acetate and 18F-FDG PET imaging analyses along with neuropsychological assessments from 11 AD patients and 10 healthy control subjects. We demonstrate that reactive astrocytes excessively absorb acetate through elevated monocarboxylate transporter-1 (MCT1) in rodent models of both reactive astrogliosis and AD. The elevated acetate uptake is associated with reactive astrogliosis and boosts the aberrant astrocytic GABA synthesis when amyloid-ß is present. The excessive astrocytic GABA subsequently suppresses neuronal activity, which could lead to glucose uptake through decreased glucose transporter-3 in the diseased brains. We further demonstrate that 11C-acetate uptake was significantly increased in the entorhinal cortex, hippocampus and temporo-parietal neocortex of the AD patients compared to the healthy controls, while 18F-FDG uptake was significantly reduced in the same regions. Additionally, we discover a strong correlation between the patients' cognitive function and the PET signals of both 11C-acetate and 18F-FDG. We demonstrate the potential value of PET imaging with 11C-acetate and 18F-FDG by visualizing reactive astrogliosis and the associated neuronal glucose hypometablosim for AD patients. Our findings further suggest that the acetate-boosted reactive astrocyte-neuron interaction could contribute to the cognitive decline in AD.

11 C-Acetate PET/CT Detects Reactive Astrogliosis Helping Glioma Classification.

PURPOSE: 11 C-acetate ( 11 C-ACE) uptake on PET/CT was recently discovered to represent reactive astrocytes in the tumor microenvironment. This study aimed at evaluating the role of 11 C-ACE PET/CT as an imaging biomarker of reactive astrogliosis in characterizing different types of gliomas. METHODS: In this prospective study, a total of 182 patients underwent 11 C-ACE PET/CT before surgery. The ratio of SUV max of a glioma to the SUV mean of the contralateral choroid plexus ( 11 C-ACE TCR) on PET/CT was calculated. 11 C-ACE TCRs were compared with the World Health Organization grades and isocitrate dehydrogenase 1 ( IDH1 ) mutation status. Grade 2 was considered low-grade tumor, and grades 3 and 4 were considered high-grade tumors. RESULTS: The median 11 C-ACE TCR was significantly higher in IDH1 wild-type (wt) tumors (n = 91) than in IDH1 -mutant (mt) tumors (n = 91) (2.38 vs 1.30, P < 0.001). Of the 91 IDH1 -mt tumors, there were no differences in the median 11 C-ACE TCRs between oligodendrogliomas (ODs) and astrocytic tumors (1.40 vs 1.20, P > 0.05). In grading low- versus high-grade gliomas, the receiver operating characteristic curve analyses showed a higher area under the curve (0.951) in IDH1 -wt tumors than in IDH1 -mt tumors (0.783, P = 0.002). Grade 2 ODs were well differentiated from high-grade gliomas. The 11 C-ACE TCR of grade 3 ODs was significantly lower than that of IDH1 -wt glioblastomas. CONCLUSIONS: High 11 C-ACE uptake is associated with high-grade IDH1 -wt tumors, thus facilitating differentiation from high-grade IDH1-mt and low-grade gliomas. In particular, low 11 C-ACE uptake in ODs is advantageous in overcoming the limitation of radiolabeled amino acid tracers.

The Quinazoline Otaplimastat (SP-8203) Reduces the Hemorrhagic Transformation and Mortality Aggravated after Delayed rtPA-Induced Thrombolysis in Cerebral Ischemia.

Acute ischemic stroke is the leading cause of morbidity and mortality worldwide. Recombinant tissue plasminogen activator (rtPA) is the only agent clinically approved by FDA for patients with acute ischemic stroke. However, delayed treatment of rtPA (e.g., more than 3 h after stroke onset) exacerbates ischemic brain damage by causing intracerebral hemorrhage and increasing neurotoxicity. In the present study, we investigated whether the neuroprotant otaplimastat reduced delayed rtPA treatment-evoked neurotoxicity in male Sprague Dawley rats subjected to embolic middle cerebral artery occlusion (eMCAO). Otaplimastat reduced cerebral infarct size and edema and improved neurobehavioral deficits. In particular, otaplimastat markedly reduced intracerebral hemorrhagic transformation and mortality triggered by delayed rtPA treatment, consequently extending the therapeutic time window of rtPA. We further found that ischemia-evoked extracellular matrix metalloproteases (MMPs) expression was closely correlated with cerebral hemorrhagic transformation and brain damage. In ischemic conditions, delayed rtPA treatment further increased brain injury via synergistic expression of MMPs in vascular endothelial cells. In oxygen-glucose-deprived endothelial cells, otaplimastat suppressed the activity rather than protein expression of MMPs by restoring the level of tissue inhibitor of metalloproteinase (TIMP) suppressed in ischemia, and consequently reduced vascular permeation. This paper shows that otaplimastat under clinical trials is a new drug which can inhibit stroke on its own and extend the therapeutic time window of rtPA, especially when administered in combination with rtPA.

Glucose Loading Enhances the Value of 18F-FDG PET/CT for the Characterization and Delineation of Cerebral Gliomas.

This study aimed to assess how to enhance the value of 18F-Fluorodeoxyglucose (FDG) PET/CTs for glioma grading and better delineation of the tumor boundary by glucose loading. In mouse models of brain tumor using U87MG cells, 18F-FDG-PET images were obtained after fasting and after glucose loading. There was a significant difference in the tumor-to-normal cortex-uptake ratio (TNR) between the fasting and glucose-loading scans. 14C-2-Deoxy-D-glucose (14C-DG) uptake was measured in vitro using U87MG, U373MG and primary neurons cultured with different concentrations of glucose. The tumor-to-neuron ratio of 14C-DG uptake increased with up to 10 mM of glucose. Finally, 10 low-grade and 17 high-grade glioma patients underwent fasting and glucose loading 18F-FDG PET/CT and the TNR was compared between scans. The effect of glucose loading was significant in high-grade but not in low-grade gliomas. The receiver operating characteristic curve analyses with a cut-off TNR of 0.81 showed a higher area under the curve after glucose loading than fasting for differentiating low-grade versus high-grade gliomas. In addition, the glucose loading PET/CT was more useful than the fasting PET/CT for the discrimination of oligodendrogliomas from IDH-wildtype glioblastomas. Glucose loading resulted in a greater reduction in 18F-FDG uptake in the normal cortex than in tumors, which increases the usefulness of 18F-FDG PET/CT for grading.

Modulation of SIRT3 expression through CDK4/6 enhances the anti-cancer effect of sorafenib in hepatocellular carcinoma cells.

BACKGROUND: Hepatocellular carcinoma (HCC) is the leading cause of cancer-related deaths worldwide. The only drug currently approved for clinical use in the treatment of advanced HCC is sorafenib. However, many patients with HCC show reduced sensitivity to sorafenib during treatment. SIRT3, a member of the mammalian sirtuin family, is a tumor suppressor in certain tumor types. However, only few studies have investigated the effects of SIRT3 on tumor prognosis and sorafenib sensitivity in patients with HCC. Here, we aimed to investigate the correlation between SIRT3 expression and glucose metabolism and proliferation in HCC and discover effective compounds that increase endogenous SIRT3 modulation effect of sorafenib. METHODS: To determine the correlation between SIRT3 and glucose related proteins, immunostaining was performed with liver cancer tissue using various antibodies. To investigate whether the expression of SIRT3 in HCC is related to the resistance to sorafenib, we treated sorafenib after the modulation of SIRT3 levels in HCC cell lines (overexpression in Huh7, knockdown in HepG2). We also employed PD0332991 to modulate the SIRT3 expression in HCC cell and conducted functional assays. RESULTS: SIRT3 expression was downregulated in high glycolytic and proliferative HCC cells of human patients, xenograft model and HCC cell lines. Moreover, SIRT3 expression was downregulated after sorafenib treatment, resulting in reduced drug sensitivity in HCC cell lines. To enhance the anti-tumor effect of sorafenib, we employed PD0332991 (CDK4/6-Rb inhibitor) based on the correlation between SIRT3 and phosphorylated retinoblastoma protein in HCC. Notably, combined treatment with sorafenib and PD0332991 showed an enhancement of the anti-tumor effect in HCC cells. CONCLUSIONS: Our data suggest that the modulation of SIRT3 by CDK4/6 inhibition might be useful for HCC therapy together with sorafenib, which, unfortunately, has limited efficacy and whose use is often associated with drug resistance.

The critical role of glucose deprivation in epithelial-mesenchymal transition in hepatocellular carcinoma under hypoxia.

Imaging with 18F-fluorodeoxyglucose (FDG) positron emission tomography/computed tomography (PET/CT) is used to determine sites of abnormal glucose metabolism to predict high tumor grade, metastasis, and poor patient survival. However, not all tumors with increased 18F-FDG uptake show aggressive tumor biology, as evident from the moderate correlation between metastasis and high FDG uptake. We hypothesized that metastasis is likely attributable to the complexity and heterogeneity of the cancer microenvironment. To identify the cancer microenvironment that induces the epithelial-mesenchymal transition (EMT) process, tumor areas of patients with HCC were analyzed by immunostaining. Our data demonstrated the induction of EMT process in HCC cells with low proliferation under hypoxic conditions. To validate our finding, among HCC cell lines, HepG2 cells with highly increased expression of HIF1α under hypoxia were employed in vitro and in vivo. Major changes in EMT-associated protein expression, such as the up-regulation of N-cadherin and snail/slug are associated with decreased proliferation-related protein (PCNA) caused by glucose deprivation under hypoxia. Indeed, PCNA knockdown-HepG2 cells under hypoxia showed the induction of more EMT process compare to the control. Thus, HCC cells with low proliferative potential under glucose-deprived and hypoxic conditions show high probability for induced EMT process and promote cell invasion. This study investigates reasons as to why an EMT process cannot fully be predicted. Our observations indicate that rather than analyzing a single factor, an integrated analysis of hypoxia with low glucose metabolism and low cell proliferation might be helpful to predict the potential impact on induction of EMT process and promotion of cell invasion.

Jak kinase 3 signaling in microgliogenesis from the spinal nestin+ progenitors in both development and response to injury.

During spinal cord development, endogenous progenitors expressing nestin can migrate into the target and differentiate into neurons and other glial cells. Microglial cells can also be derived from nestin progenitor cells, even in the adult brain. Knockdown of Jak kinase 3 (Jak3) signaling can increase neurogenesis with longer neurite outgrowth in cortical progenitor cells. This study investigated the effect of Jak3 signaling on differentiation from nestin progenitor cells using E13.5 spinal progenitor cell cultures. In growth factors-enriched culture, developing neurons could not survive after several days and also a significant proportion of nestin-expressing cells transformed into ameboid Iba1 microglial cells, which increased exponentially after 5 days. This microgliogenesis was predominantly regulated by Jak3 signaling, which was confirmed by transcription factors responsible for microgliogenesis, and microglial migration and phagocytosis, such as Pu.1, Irf8, and Runx1. Jak3 inhibition also significantly increased the Tuj1 growing neurites with little microglial activation. These results indicated that neuronal and microglial cell differentiation was regulated primarily by Jak3 signaling and the developing neurons and neurite outgrowth might also be regulated by Jak3-dependent microglial activity.

Effect of Continuous-Wave Low-Intensity Ultrasound in Inflammatory Resolution of Arthritis-Associated Synovitis.

BACKGROUND: Low-intensity ultrasound (LIUS) can reduce pain and improve function in arthritic joints. Neutrophils are first-line actors in host defense that recruit macrophages. Dead neutrophils are removed during resolution of inflammation. Delayed neutrophil clearance can lead to extended inflammation or even chronic autoimmune disease. Although neutrophil extracellular traps (NETs) in arthritic tissue are involved in the pathogenesis of arthritis, their functional role has not been clarified. OBJECTIVES: This study aimed to investigate the effect of LIUS on synovial inflammation and its resolution via neutrophil clearance. METHODS: Synovitis was induced by intra-articular injection of complete Freund's adjuvant (CFA) into the left knee joint of 58 adult male Sprague-Dawley rats. Low-intensity ultrasound (1 MHz, 200 mW/cm(2)) was applied for 10 minutes daily. Neutrophil clearance was assessed with the expression of myeloperoxidase (MPO). In addition, terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining and NET formation in the synovium were observed. In neutrophil and macrophage cultures from peripheral blood, the effect of NET clearance by LIUS was investigated. RESULTS: In CFA-induced synovitis, MPO-positive neutrophils peaked after 2 to 3 days, filling the inflammatory core. Monocytes and macrophages in the periphery later infiltrated the core and were reduced thereafter. Low-intensity ultrasound reduced synovial hyperplasia and induced earlier MPO clearance. Neutrophils in the core of the inflamed synovium exhibited NET formation, which LIUS increased. Low-intensity also induced NETs in peripheral polymorphonuclear cells in an intensity-dependent manner and potentiated phorbol myristate acetate (PMA)-induced NETosis. The PMA-induced NETs were cleared by macrophages; clearance was enhanced by LIUS. LIMITATIONS: The effect of LIUS on CFA-induced inflammation was observed only during the acute phase. Although the effect of LIUS on NETosis in the in vitro neutrophil culture system was clear, the in vivo NETosis cannot be quantified. CONCLUSIONS: Neutrophil extracellular traps act in inflammatory synovitis, and LIUS enhanced the NETs and resulted in neutrophil clearance by enhancing the phagocytosis of macrophages, which might be a factor underlying the therapeutic effect of LIUS in arthritic synovium.

Seizure susceptibility in immature brain due to lack of COX-2-induced PGF2α.

The immature brain is prone to seizure; however, the mechanism underlying this vulnerability has not been clarified. Febrile seizure is common in young children, and the use of non-steroidal anti-inflammatory drugs for febrile seizure is not recommended. In previous studies, we established that prostaglandin (PG) F2α, a product of cyclooxygenase (COX), acts as an endogenous anticonvulsant in the adult mouse. Therefore, we assumed that COX-2 activity was involved with seizure susceptibility in early life. In the present study, immature mice (postnatal day 9) were far more prone to kainic acid (KA)-induced seizures than mature mice (after postnatal day 35). Seizure activity began later in immature mice, but was more severe and was unaffected by a potent COX inhibitor, indomethacin; in contrast, indomethacin aggravated seizure activity in mature mice. Immature mouse brains exhibited little basal COX-2 expression and little KA-induced COX-2 induction, while KA-induced COX-2 expression and PGF2α release were prominent in mature brains. During brain development, COX expression was increased and glycosylated in an age-dependent manner, which was necessary for COX enzyme activity. Intracisternal PGF2α administration also reduced KA-induced seizure activity and mortality. Taken together, low COX activity and the resulting deficiency of PGF2α may be an essential cause of increased seizure susceptibility in the immature brain.

Differential regulation of proliferation and differentiation in neural precursor cells by the Jak pathway.

Neuronal precursor cells (NPCs) are temporally regulated and have the ability to proliferate and differentiate into mature neurons, oligodendrocytes, and astrocytes in the presence of growth factors (GFs). In the present study, the role of the Jak pathway in brain development was investigated in NPCs derived from neurosphere cultures using Jak2 and Jak3 small interfering RNAs and specific inhibitors. Jak2 inhibition profoundly decreased NPC proliferation, preventing further differentiation into neurons and glial cells. However, Jak3 inhibition induced neuronal differentiation accompanied by neurite growth. This phenomenon was due to the Jak3 inhibition-mediated induction of neurogenin (Ngn)2 and NeuroD in NPCs. Jak3 inhibition induced NPCs to differentiate into scattered neurons and increased the expression of Tuj1, microtubule associated protein 2 (MAP2), Olig2, and neuroglial protein (NG)2, but decreased glial fibrillary acidic protein (GFAP) expression, with predominant neurogenesis/polydendrogenesis compared with astrogliogenesis. Therefore, Jak2 may be important for NPC proliferation and maintenance, whereas knocking-down of Jak3 signaling is essential for NPC differentiation into neurons and oligodendrocytes but does not lead to astrocyte differentiation. These results suggest that NPC proliferation and differentiation are differentially regulated by the Jak pathway.

Involvement of endogenous prostaglandin F2alpha on kainic acid-induced seizure activity through FP receptor: the mechanism of proconvulsant effects of COX-2 inhibitors.

COX-2 and prostaglandins (PGs) might play important roles in epilepsy. In kainic acid-induced seizures, the brain largely increases PGD(2), first from COX-1 and later COX-2-induced PGF(2alpha). Pre-treatment with COX-2 inhibitors such as indomethacin, nimesulide, and celecoxib is known to aggravate kainic acid (KA)-induced seizure activity. However it is not known whether the proconvulsant effect of those non-steroidal anti-inflammatory drugs (NSAIDs) is due to changes in endogenous prostaglandins (PGs), or what types of PGs are involved. The purpose of this study was to determine the effect of intracisternally administered PGs on KA-induced seizures aggravated by pre- or post-treatment with COX-2 inhibitors. Systemic KA injection (10 mg/kg i.p.) in mice evoked mild seizure activity within 15 min. PGs were administrated intracisternally 20 min prior to KA administration. COX inhibitors (indomethacin, nimesulide, and ketoprofen, 10 mg/kg i.p.) were injected 1 h before or 15 min after KA. An additional COX-2 inhibitor, celecoxib, was administered orally. Intracisternally administered PGF(2alpha) (700 ng), but not PGD(2) (700 ng) or PGE(2) (700 ng) completely alleviated KA-induced seizures potentiated by COX-2 inhibitors, and also reduced KA-induced hippocampal neuronal death aggravated by indomethacin. PGF(2alpha) alone did not affect KA-induced seizures. However, an FP receptor antagonist, AL 8810 (10 or 50 ng) which is an 11beta-fluoro analogue of PGF(2alpha) potentiated KA-induced seizure activity dose-dependently. In summary, pre- or post-treatment with COX-2 inhibitors aggravates KA-induced seizures, which suggests to change the endogenous PGF(2alpha). Seizure-induced PGF(2alpha) might act as an endogenous anticonvulsant through FP receptors.