A dopamine D1-like receptor-specific agonist improves the survival of septic mice.

In this study, a murine sepsis model was developed using the cecum ligation and puncture (CLP) technique. The expression of the proinflammatory cytokines tumor necrosis factor alpha (TNF-α) and interleukin-1ß (IL-1ß) in the brain increased 6 h after CLP but decreased 24 h later when elevated endogenous dopamine levels in the brain were sustained. Methyl-4-phenyl-1,2,3,6-tetrahydropyridine hydrochloride reduced dopamine levels in the striatum and increased mortality in septic mice. Dopamine D1-like receptors were significantly expressed in the brain, but not in the lungs. Intraperitoneally administered SKF-81297 (SKF), a blood-brain barrier-permeable D1-like receptor agonist, prevented CLP-induced death of septic mice with ameliorated acute lung injury and cognitive dysfunction and suppressed TNF-α and IL-1ß expression. The D1-like receptor antagonist SCH-23390 abolished the anti-inflammatory effects of SKF. These data suggest that D1-like receptor-mediated signals in the brain prevent CLP-induced inflammation in both the brain and the periphery.

Simultaneous disturbance of NHE1 and LOXL2 decreases tumorigenicity of head and neck squamous cell carcinoma.

OBJECTIVE: Although there have been brilliant advancements in the practical application of therapies targeting immune checkpoints, achieving success in targeting the microenvironment remains elusive. In this study, we aimed to address this gap by focusing on Na+ / H+ exchanger 1 (NHE1) and Lysyl Oxidase Like 2 (LOXL2), which are upregulated in head and neck squamous cell carcinoma (HNSCC) cells. METHODS: The malignancy of a metastatic human HNSCC cell line was assessed in a mouse tongue cancer xenograft model by knocking down (KD) NHE1, responsible for regulating intracellular pH, and LOXL2, responsible for extracellular matrix (ECM) reorganization via cross-linking of ECM proteins. In addition to assessing changes in PD-L1 levels and collagen accumulation following knockdown, the functional status of the PD-L1 / PD-1 immune checkpoint was examined through co-culture with NK92MI, a PD-1 positive phagocytic human Natural Killer (NK) cell line. RESULTS: The tumorigenic potential of each single KD cell line was similar to that of the control cells, whereas the potential was attenuated in cells with simultaneous KD of both factors (double knockdown [dKD]). Additionally, we observed decreased PD-L1 levels in NHE1 KD cells and compromised collagen accumulation in LOXL2 KD and dKD cells. NK92MI cells exhibited phagocytic activity toward HNSCC cells in co-culture, and the number of remaining dKD cells after co-culture was the lowest in comparison to the control and single KD cells. CONCLUSION: This study demonstrated the possibility of achieving efficient anti-tumor effects by simultaneously disturbing multiple factors involved in the modification of the tumor microenvironment.

Berberine as a potential enhancer for 5-ALA-mediated fluorescence in glioblastoma: increasing detectability of infiltrating glioma stem cells to optimize 5-ALA-guided surgery.

OBJECTIVE: The prognosis of glioblastoma (GBM) correlates with residual tumor volume after surgery. In fluorescence-guided surgery, 5-aminolevulinic acid (ALA) has been used to maximize resection while avoiding neurological morbidity. However, not all tumor cells, particularly glioma stem cells (GSCs), display 5-ALA-mediated protoporphyrin IX (PpIX) fluorescence (5-ALA fluorescence). The authors searched for repositioned drugs that affect mitochondrial functions and energy metabolism, identifying berberine (BBR) as a potential enhancer of 5-ALA fluorescence. In this study, they investigated whether BBR can enhance 5-ALA fluorescence in GSCs and whether BBR can be applied to clinical practice as a 5-ALA fluorescence enhancer. METHODS: The effects of BBR on 5-ALA fluorescence in glioma and GSCs were evaluated by flow cytometry (fluorescence-activated cell sorting [FACS]) analysis. As 5-ALA is metabolized for heme synthesis, the effects of BBR on mRNA expressions of 7 enzymes in the heme-synthesis pathway were analyzed. Enzymes showing significantly higher expression than control in all cells were identified and protein analysis was performed. To examine clinical availability, the detectability and cytotoxicity of BBR in tumor-transplanted mice were analyzed. RESULTS: Fluorescence microscopy revealed much more intense 5-ALA fluorescence in both GSCs and non-stem cells with 5-ALA and BBR than with 5-ALA alone. FACS showed that BBR greatly enhanced 5-ALA fluorescence compared with 5-ALA alone, and enhancement was much higher for GSCs than for glioma cells. Among the 7 enzymes examined, BBR upregulated mRNA expressions of ALA synthetase 1 (ALAS1) more highly in all cells, and activated ALAS1 through deregulating ALAS1 activity inhibited by the negative feedback of heme. An in vivo study showed that 5-ALA fluorescence with 5-ALA and BBR was significantly stronger than with 5-ALA alone, and the sensitivity and specificity of BBR-enhanced fluorescence were both 100%. In addition, BBR did not show any cytotoxicity for normal brain tissue surrounding the tumor mass. CONCLUSIONS: BBR enhanced 5-ALA-mediated PpIX fluorescence by upregulating and activating ALAS1 through deregulation of negative feedback inhibition by heme. BBR is a clinically used drug with no side effects. BBR is expected to significantly augment fluorescence-guided surgery and photodynamic therapy.

A Narrative Review on CD44's Role in Glioblastoma Invasion, Proliferation, and Tumor Recurrence.

High invasiveness is a characteristic of glioblastoma (GBM), making radical resection almost impossible, and thus, resulting in a tumor with inevitable recurrence. GBM recurrence may be caused by glioma stem-like cells (GSCs) that survive many kinds of therapy. GSCs with high expression levels of CD44 are highly invasive and resistant to radio-chemotherapy. CD44 is a multifunctional molecule that promotes the invasion and proliferation of tumor cells via various signaling pathways. Among these, paired pathways reciprocally activate invasion and proliferation under different hypoxic conditions. Severe hypoxia (0.5-2.5% O2) upregulates hypoxia-inducible factor (HIF)-1α, which then activates target genes, including CD44, TGF-ß, and cMET, all of which are related to tumor migration and invasion. In contrast, moderate hypoxia (2.5-5% O2) upregulates HIF-2α, which activates target genes, such as vascular endothelial growth factor (VEGF)/VEGFR2, cMYC, and cyclin D1. All these genes are related to tumor proliferation. Oxygen environments around GBM can change before and after tumor resection. Before resection, the oxygen concentration at the tumor periphery is severely hypoxic. In the reparative stage after resection, the resection cavity shows moderate hypoxia. These observations suggest that upregulated CD44 under severe hypoxia may promote the migration and invasion of tumor cells. Conversely, when tumor resection leads to moderate hypoxia, upregulated HIF-2α activates HIF-2α target genes. The phenotypic transition regulated by CD44, leading to a dichotomy between invasion and proliferation according to hypoxic conditions, may play a crucial role in GBM recurrence.

Identification of CD44 as a Reliable Biomarker for Glioblastoma Invasion: Based on Magnetic Resonance Imaging and Spectroscopic Analysis of 5-Aminolevulinic Acid Fluorescence.

Recurrent glioblastoma multiforme (GBM) is largely attributed to peritumoral infiltration of tumor cells. As higher CD44 expression in the tumor periphery correlates with higher risk of GBM invasion, the present study analyzed the relationship between CD44 expression and magnetic resonance imaging (MRI)-based invasiveness of GBM on a large scale. We also quantitatively evaluated GBM invasion using 5-aminolevulinic acid (5-ALA) spectroscopy to investigate the relationship between CD44 expression and tumor invasiveness as evaluated by intraoperative 5-ALA intensity. Based on MRI, GBM was classified as high-invasive type in 28 patients and low-invasive type in 22 patients. High-invasive type expressed CD44 at a significantly higher level than low-invasive type and was associated with worse survival. To quantitatively analyze GBM invasiveness, the relationship between tumor density in the peritumoral area and the spectroscopic intensity of 5-ALA was investigated. Spectroscopy showed that the 5-ALA intensity of infiltrating tumor cells correlated with tumor density as represented by the Ki-67 staining index. No significant correlation between CD44 and degree of 5-ALA-based invasiveness of GBM was found, but invasiveness of GBM as evaluated by 5-ALA matched the classification from MRI in all except one case, indicating that CD44 expression at the GBM periphery could provide a reliable biomarker for invasiveness in GBM.

The anti-inflammatory and anti-oxidative effect of a classical hypnotic bromovalerylurea mediated by the activation of NRF2.

The Kelch-like ECH-associated protein 1-nuclear factor erythroid 2-related factor 2 (KEAP1-NRF2) system plays a central role in redox homeostasis and inflammation control. Oxidative stress or electrophilic compounds promote NRF2 stabilization and transcriptional activity by negatively regulating its inhibitor, KEAP1. We have previously reported that bromovalerylurea (BU), originally developed as a hypnotic, exerts anti-inflammatory effects in various inflammatory disease models. However, the molecular mechanism underlying its effect remains uncertain. Herein, we found that by real-time multicolor luciferase assay using stable luciferase red3 (SLR3) and green-emitting emerald luciferase (ELuc), BU potentiates NRF2-dependent transcription in the human hepatoblastoma cell line HepG2 cells, which lasted for more than 60 h. Further analysis revealed that BU promotes NRF2 accumulation and the transcription of its downstream cytoprotective genes in the HepG2 and the murine microglial cell line BV2. Keap1 knockdown did not further enhance NRF2 activity, suggesting that BU upregulates NRF2 by targeting KEAP1. Knockdown of Nfe2l2 in BV2 cells diminished the suppressive effects of BU on the production of pro-inflammatory mediators, like nitric oxide (NO) and its synthase NOS2, indicating the involvement of NRF2 in the anti-inflammatory effects of BU. These data collectively suggest that BU could be repurposed as a novel NRF2 activator to control inflammation and oxidative stress.

Anti-inflammatory effects of dopamine on microglia and a D1 receptor agonist ameliorates neuroinflammation of the brain in a rat delirium model.

Microglia play a central role in neuroinflammatory processes by releasing proinflammatory mediators. This process is tightly regulated along with neuronal activities, and neurotransmitters may link neuronal activities to the microglia. In this study, we showed that primary cultured rat microglia express the dopamine (DA) D1 receptor (D1R) and D4R, but not D2R, D3R, or D5R. In response to a D1R-specific agonist SKF-81297 (SKF), the cultured microglia exhibited increased intracellular cAMP levels. DA and SKF suppressed lipopolysaccharide (LPS)-induced expression of interleukin-1ß (IL-1ß) and tumor necrosis α (TNFα) in cultured microglia. Microglia in the normal mature rat prefrontal cortex (PFC) were sorted and significant expression of D1R, D2R, and D4R was observed. A delirium model was established by administering LPS intraperitoneally to mature male Wistar rats. The model also displayed sleep-wake disturbances as revealed by electroencephalogram and electromyogram recordings as well as increased expression of IL-1ß and TNFα in the PFC. DA levels were increased in the PFC 21 h after LPS administration. Increased cytokine expression was observed in sorted microglia from the PFC of the delirium model; however, TNFα, but not IL-1ß expression, was abruptly decreased 21 h after LPS administration in the delirium model, whereas DA levels were increased. A D1R antagonist SCH23390 partially abolished the TNFα expression change. This suggests that endogenous DA may play a role in suppressing neuroinflammation. Administration of the DA precursor L-DOPA or SKF to the delirium model rats inhibited the expression of IL-1ß and TNFα. The simultaneous administration of clozapine, a D4R antagonist, strengthened the suppressive effects of L-DOPA. These results suggest that D1R mediates the suppressive effects of LPS-induced neuroinflammation, in which microglia may play an important role. Agonists for D1R may be effective for treating delirium.

Quantitative measurement of peritumoral concentrations of glutamate, N-acetyl aspartate, and lactate on magnetic resonance spectroscopy predicts glioblastoma-related refractory epilepsy.

BACKGROUND: Increased extracellular glutamate is known to cause epileptic seizures in patients with glioblastoma (GBM). However, predicting whether the seizure will be refractory is difficult. The present study investigated whether evaluation of the levels of various metabolites, including glutamate, can predict the occurrence of refractory seizure in GBM by quantitative measurement of metabolite concentrations on magnetic resonance spectroscopy (MRS). METHODS: Forty patients were treated according to the same treatment protocol for primary GBM at Ehime University Hospital between April 2017 and July 2021. Of these patients, 23 underwent MRS to determine concentrations of metabolites, including glutamate, N-acetylaspartate, creatine, and lactate, in the tumor periphery by applying LC-Model. The concentration of each metabolite was expressed as a ratio to creatine concentration. Patients were divided into three groups: Type A, patients with no seizures; Type B, patients with seizures that disappeared after treatment; and Type C, patients with seizures that remained unrelieved or appeared after treatment (refractory seizures). Relationships between concentrations of metabolites and seizure types were investigated. RESULTS: In 23 GBMs, seizures were confirmed in 11 patients, including Type B in four and Type C in seven. Patients with epilepsy (Type B or C) showed significantly higher glutamate and N-acetylaspartate values than did non-epilepsy patients (Type A) (p < 0.05). No significant differences in glutamate or N-acetylaspartate levels were seen between Types B and C. Conversely, Type C showed significantly higher concentrations of lactate than did Type B (p = 0.001). Cutoff values of lactate-to-creatine, glutamate-to-creatine, and N-acetylaspartate-to-creatine ratios for refractory seizure were > 1.25, > 1.09, and > 0.88, respectively. CONCLUSIONS: Extracellular concentrations of glutamate, N-acetylaspartate, and lactate in the tumor periphery were significantly elevated in patients with GBM with refractory seizures. Measurement of these metabolites on MRS may predict refractory epilepsy in such patients and could be an indicator for continuing the use of antiepileptic drugs.

Microglial re-modeling contributes to recovery from ischemic injury of rat brain: A study using a cytokine mixture containing granulocyte-macrophage colony-stimulating factor and interleukin-3.

Ischemic stroke is a leading cause of mortality and permanent disability. Chronic stroke lesions increase gradually due to the secondary neuroinflammation that occurs following acute ischemic neuronal degeneration. In this study, the ameliorating effect of a cytokine mixture consisting of granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin (IL)-3 was evaluated on ischemic brain injury using a rat stroke model prepared by transient middle cerebral artery occlusion (tMCAO). The mixture reduced infarct volume and ameliorated ischemia-induced motor and cognitive dysfunctions. Sorted microglia cells from the ischemic hemisphere of rats administered the mixture showed reduced mRNA expression of tumor necrosis factor (TNF)-α and IL-1ß at 3 days post-reperfusion. On flow cytometric analysis, the expression of CD86, a marker of pro-inflammatory type microglia, was suppressed, and the expression of CD163, a marker of tissue-repairing type microglia, was increased by the cytokine treatment. Immunoblotting and immunohistochemistry data showed that the cytokines increased the expression of the anti-apoptotic protein Bcl-xL in neurons in the ischemic lesion. Thus, the present study demonstrated that cytokine treatment markedly suppressed neurodegeneration during the chronic phase in the rat stroke model. The neuroprotective effects may be mediated by phenotypic changes of microglia that presumably lead to increased expression of Bcl-xL in ischemic lesions, while enhancing neuronal survival.

Space Radiation Dosimetry at the Exposure Facility of the International Space Station for the Tanpopo Mission.

Radiation dosimetry was carried out at the exposure facility (EF) and the pressurized module (PM) of the Japanese Kibo module installed in the International Space Station as one study on environmental monitoring for the Tanpopo mission. Three exposure panels and three references including biological and organic samples and luminescence dosimeters were launched to obtain data for different exposure durations during 3 years from May 2015 to July 2018. The dosimeters were equipped with additional shielding materials (0.55, 2.95, and 6.23 g/cm2 mass thickness). The relative dose variation, as a function of shielding mass thickness, was observed and compared with Monte Carlo simulations with respect to galactic cosmic rays (GCRs) and typical solar energetic particles (SEPs). The mean annual dose rates were DEF = 231 ± 5 mGy/year at the EF and DPM = 82 ± 1 mGy/year at the PM during the 3 years. The PM is well shielded, and the GCR simulation indicated that the measured mean dose reduction ratio inside the module (DPM/DEF = 0.35) required ∼26 g/cm2 additional shielding mass thickness. Observed points of the dose reduction tendency could be explained by the energy ranges of protons (10-100 MeV), where the protons passed through, or were absorbed in, the shielding materials of different mass thickness that surrounded dosimeters.

Chloride intracellular channel protein 2 is secreted and inhibits MMP14 activity, while preventing tumor cell invasion and metastasis.

The abilities to invade surrounding tissues and metastasize to distant organs are the most outstanding features that distinguish malignant from benign tumors. However, the mechanisms preventing the invasion and metastasis of benign tumor cells remain unclear. By using our own rat distant metastasis model, gene expression of cells in primary tumors was compared with that in metastasized tumors. Among many distinct gene expressions, we have focused on chloride intracellular channel protein 2 (CLIC2), an ion channel protein of as-yet unknown function, which was predominantly expressed in the primary tumors. We created CLIC2 overexpressing rat glioma cell line and utilized benign human meningioma cells with naturally high CLIC2 expression. CLIC2 was expressed at higher levels in benign human brain tumors than in their malignant counterparts. Moreover, its high expression was associated with prolonged survival in the rat metastasis and brain tumor models as well as with progression-free survival in patients with brain tumors. CLIC2 was also correlated with the decreased blood vessel permeability likely by increased contents of cell adhesion molecules. We found that CLIC2 was secreted extracellularly, and bound to matrix metalloproteinase (MMP) 14. Furthermore, CLIC2 prevented the localization of MMP14 in the plasma membrane, and inhibited its enzymatic activity. Indeed, overexpressing CLIC2 and recombinant CLIC2 protein effectively suppressed malignant cell invasion, whereas CLIC2 knockdown reversed these effects. Thus, CLIC2 suppress invasion and metastasis of benign tumors at least partly by inhibiting MMP14 activity.

Prediction of Glioma Stemlike Cell Infiltration in the Non-Contrast-Enhancing Area by Quantitative Measurement of Lactate on Magnetic Resonance Spectroscopy in Glioblastoma.

BACKGROUND: We previously reported that glioma stemlike cells (GSCs) exist in the area of the tumor periphery showing no gadolinium enhancement on magnetic resonance imaging. In the present work, we analyzed glucose metabolism to investigate whether lactate could be predictive of tumor invasiveness and of use in detection of the tumor invasion area in glioblastoma multiforme (GBM). METHODS: The expression of lactate dehydrogenase A (LDH-A) and pyruvate dehydrogenase (PDH) was investigated in 20 patients. In GSC lines, LDH-A and PDH expression also was examined in parallel to assessments of mitochondrial respiration. We then investigated the relationship between lactate/creatine ratios in the tumor periphery measured by magnetic resonance spectroscopy, using learning-compression-model algorithms and phenotypes of GBMs. RESULTS: In 20 GBMs, high-invasive GBM expressed LDH-A at significantly higher expression than did low-invasive GBM, whereas low-invasive GBM showed significantly higher expression of PDH than did high-invasive GBM. The highly invasive GSC line showed higher expression of LDH-A and lower expression of PDH compared with low-invasive GSC lines. The highly invasive GSC line also showed the lowest consumption of oxygen and the lowest production of adenosine triphosphate. Lactate levels, as measured by magnetic resonance spectroscopy, showed a significant positive correlation with LDH-A transcript levels, permitting classification of the GBMs into high-invasive and low-invasive phenotypes based on a cutoff value of 0.66 in the lactate/creatine ratio. CONCLUSIONS: In the tumor periphery area of the highly invasive GBM, aerobic glycolysis was the predominant pathway for glucose metabolism, resulting in the accumulation of lactate. The level of lactate may facilitate prediction of the tumor-infiltrating area on GBM.

Hypoxia-induced phenotypic transition from highly invasive to less invasive tumors in glioma stem-like cells: Significance of CD44 and osteopontin as therapeutic targets in glioblastoma.

The poor prognosis of glioblastoma multiforme (GBM) is primarily due to highly invasive glioma stem-like cells (GSCs) in tumors. Upon GBM recurrence, GSCs with highly invasive and highly migratory activities must assume a less-motile state and proliferate to regenerate tumor mass. Elucidating the molecular mechanism underlying this transition from a highly invasive phenotype to a less-invasive, proliferative tumor could facilitate the identification of effective molecular targets for treating GBM. Here, we demonstrate that severe hypoxia (1% O2) upregulates CD44 expression via activation of hypoxia-inducible factor (HIF-1α), inducing GSCs to assume a highly invasive tumor. In contrast, moderate hypoxia (5% O2) upregulates osteopontin expression via activation of HIF-2α. The upregulated osteopontin inhibits CD44-promoted GSC migration and invasion and stimulates GSC proliferation, inducing GSCs to assume a less-invasive, highly proliferative tumor. These data indicate that the GSC phenotype is determined by interaction between CD44 and osteopontin. The expression of both CD44 and osteopontin is regulated by differential hypoxia levels. We found that CD44 knockdown significantly inhibited GSC migration and invasion both in vitro and in vivo. Mouse brain tumors generated from CD44-knockdown GSCs exhibited diminished invasiveness, and the mice survived significantly longer than control mice. In contrast, siRNA-mediated silencing of the osteopontin gene decreased GSC proliferation. These results suggest that interaction between CD44 and osteopontin plays a key role in tumor progression in GBM; inhibition of both CD44 and osteopontin may represent an effective therapeutic approach for suppressing tumor progression, thus resulting in a better prognosis for patients with GBM.

CD44 expression in the tumor periphery predicts the responsiveness to bevacizumab in the treatment of recurrent glioblastoma.

Antiangiogenic therapy with bevacizumab (Bev), a monoclonal antibody targeting vascular endothelial growth factor (VEGF), is a common treatment for recurrent glioblastoma (GBM), but its survival benefit is limited. Resistance to Bev is thought to be a major cause of ineffectiveness on Bev therapy. To optimize Bev therapy, identification of a predictive biomarker for responsiveness to Bev is required. Based on our previous study, we focused on the expression and functions of CD44 and VEGF in the Bev therapy. Here, we analyze a relationship between CD44 expression and responsiveness to Bev and elucidate the role of CD44 in anti-VEGF therapy. CD44 and VEGF expression in the tumor core and periphery of 22 GBMs was examined, and the relationship between expression of these molecules and progression-free time on Bev therapy was analyzed. The degree of CD44 expression in the tumor periphery was evaluated by the ratio of the mRNA expression in the tumor periphery to that in the tumor core (P/C ratio). VEGF expression was evaluated by the amount of the mRNA expression in the tumor periphery. To elucidate the roles of CD44 in the Bev therapy, in vitro and in vivo studies were performed using glioma stem-like cells (GSCs) and a GSC-transplanted mouse xenograft model, respectively. GBMs expressing high P/C ratio of CD44 were much more refractory to Bev than those expressing low P/C ratio of CD44, and the survival time of the former was much shorter than that of the latter. In vitro inhibition of VEGF with siRNA or Bev-activated CD44 expression and increased invasion of GSCs. Bev showed no antitumor effects in mice transplanted with CD44-overexpressing GSCs. The P/C ratio of CD44 expression may become a useful biomarker predicting responsiveness to Bev in GBM. CD44 reduces the antitumor effect of Bev, resulting in much more highly invasive tumors.

Microglial metabolic disturbances and neuroinflammation in cerebral infarction.

Cerebral ischemia/reperfusion injury activates microglia, resident immune cells in the brain, and allows the infiltration of circulating immune cells into the ischemic lesions. Microglia play both exacerbating and protective roles in pathological processes and are thus often referred to as "double-edged swords." In ischemic brains, blood-borne macrophages play a role that is distinct from that of resident activated microglia. Recently, the metabolic alteration of immune cells in the pathogenesis of inflammatory disorders including cerebral infarction has become a critical target for investigation. We begin this review by describing the multifaceted functions of microglia in cerebral infarction. Next, we focus on the metabolic alterations that occur in microglia during pathological processes. We also discuss morphological changes that take place in the mitochondria, leading to functional disturbances, accompanied by alterations in microglial function. Moreover, we describe the involvement of the reactive oxygen species that are produced during aberrant metabolic activity. Finally, we discuss therapeutic strategies to ameliorate aggravative changes in metabolism.

Generation of CSF1-Independent Ramified Microglia-Like Cells from Leptomeninges In Vitro.

Although del Río-Hortega originally reported that leptomeningeal cells are the source of ramified microglia in the developing brain, recent views do not seem to pay much attention to this notion. In this study, in vitro experiments were conducted to determine whether leptomeninges generate ramified microglia. The leptomeninges of neonatal rats containing Iba1+ macrophages were peeled off the brain surface. Leptomeningeal macrophages strongly expressed CD68 and CD163, but microglia in the brain parenchyma did not. Leptomeningeal macrophages expressed epidermal growth factor receptor (EGFR) as revealed by RT-PCR and immunohistochemical staining. Cells obtained from the peeled-off leptomeninges were cultured in a serum-free medium containing EGF, resulting in the formation of large cell aggregates in which many proliferating macrophages were present. In contrast, colony-stimulating factor 1 (CSF1) did not enhance the generation of Iba1+ cells from the leptomeningeal culture. The cell aggregates generated ramified Iba1+ cells in the presence of serum, which express CD68 and CD163 at much lower levels than primary microglia isolated from a mixed glial culture. Therefore, the leptomeningeal-derived cells resembled parenchymal microglia better than primary microglia. This study suggests that microglial progenitors expressing EGFR reside in the leptomeninges and that there is a population of microglia-like cells that grow independently of CSF1.

Chronic constriction injury of the sciatic nerve in rats causes different activation modes of microglia between the anterior and posterior horns of the spinal cord.

Chronic constriction injury of the sciatic nerve is frequently considered as a cause of chronic neuropathic pain. Marked activation of microglia in the posterior horn (PH) has been well established with regard to this pain. However, microglial activation in the anterior horn (AH) is also strongly induced in this process. Therefore, in this study, we compared the differential activation modes of microglia in the AH and PH of the lumbar cord 7 days after chronic constriction injury of the left sciatic nerve in Wistar rats. Microglia in both the ipsilateral AH and PH demonstrated increased immunoreactivity of the microglial markers Iba1 and CD11b. Moreover, abundant CD68+ phagosomes were observed in the cytoplasm. Microglia in the AH displayed elongated somata with tightly surrounding motoneurons, whereas cells in the PH displayed a rather ameboid morphology and were attached to myelin sheaths rather than to neurons. Microglia in the AH strongly expressed NG2 chondroitin sulfate proteoglycan. Despite the tight attachment to neurons in the AH, a reduction in synaptic proteins was not evident, suggesting engagement of the activated microglia in synaptic stripping. Myelin basic protein immunoreactivity was observed in the phagosomes of activated microglia in the PH, suggesting the phagocytic removal of myelin. CCI caused both motor deficit and hyperalgesia that were evaluated by applying BBB locomotor rating scale and von Frey test, respectively. Motor defict was the most evident at postoperative day1, and that became less significant thereafter. By contrast, hyperalgesia was not severe at day 1 but it became worse at least by day 7. Collectively, the activation modes of microglia were different between the AH and PH, which may be associated with the difference in the course of motor and sensory symptoms.

Elevated exosomal lysyl oxidase like 2 is a potential biomarker for head and neck squamous cell carcinoma.

OBJECTIVES: The secretory enzyme lysyl oxidase like 2 (LOXL2) is speculated to contribute to tumor progression through its functions in the remodeling of extracellular matrix and epithelial-mesenchymal transition. We previously identified elevated expression of LOXL2 in metastatic human head and neck squamous cell carcinoma (HNSCC) cells in a mouse lymph node metastases model. Here we performed a case series study examining LOXL2 expression levels in human serum from HNSCC patients to evaluate whether LOXL2 is worth evaluation in a large cohort study. METHODS: LOXL2 protein levels in three serum samples from HNSCC patients were assessed by immunoblotting and LOXL2 tissue expression was examined in one human tongue squamous cell carcinoma (SCC) tissue by immunohistochemistry as a representative of HNSCC tissue. Serum samples were further fractionated in exosomes and supernatants by ultracentrifugation, which were then subjected to immunoblot and in vitro LOX activity analyses. Exosomal LOXL2 levels of 36 serum samples from HNSCC patients and seven healthy volunteers were measured using polymer sedimentation exosome preparation followed by ELISA measurement and subjected to statistical analyses. RESULTS: Immunoblot analyses revealed that LOXL2 was present in serum exosomal fractions from three HNSCC patients, and we observed approximately threefold higher levels of LOXL2 in HNSCC patients compared with three healthy volunteers. Immunohistochemical LOXL2 staining was detected in HNSCC cells in addition to non-cancerous lipid tissues and some muscles in human tongue HNSCC tissue. Further measurements of exosomal LOXL2 by ELISA showed over ninefold higher mean LOXL2 levels in patients compared with controls. Statistical analysis revealed a correlation between elevated serum exosomal LOXL2 levels and low-grade, but not high-grade, HNSCC. CONCLUSIONS: Our case series study that elevated serum exosomal LOXL2 levels exhibited a correlation with low-grade HNSCCs. A follow-up large cohort clinical study will be required to determine the potential clinical utility of LOXL2 as a new biomarker and/or therapy target for HNSCCs. LEVEL OF EVIDENCE: 4 Laryngoscope, 130:E327-E334, 2020.

Chloride intracellular channel protein 2 in cancer and non-cancer human tissues: relationship with tight junctions.

Chloride intracellular channel protein 2 (CLIC2) belongs to the CLIC family of conserved metazoan proteins. Although CLICs have been identified as chloride channels, they are currently considered multifunctional proteins. CLIC2 is the least studied family member. We investigated CLIC2 expression and localization in human hepatocellular carcinoma, metastatic colorectal cancer in the liver, and colorectal cancer. Significant expression of mRNAs encoding CLIC1, 2, 4, and 5 were found in the human tissues, but only CLIC2 was predominantly expressed in non-cancer tissues surrounding cancer masses. Fibrotic or dysfunctional (aspartate aminotransferase ≥40) non-cancer liver tissues and advanced stage HCC tissues expressed low levels of CLIC2. Endothelial cells lining blood vessels but not lymphatic vessels in non-cancer tissues expressed CLIC2 as well as high levels of the tight junction proteins claudins 1 and 5, occludin, and ZO-1. Most endothelial cells in blood vessels in cancer tissues had very low expressions of CLIC2 and tight junction proteins. CD31+/CD45- endothelial cells isolated from non-cancer tissues expressed mRNAs encoding CLIC2, claudin 1, occludin and ZO-1, while similar cell fractions from cancer tissues had very low expressions of these molecules. Knockdown of CLIC2 expression in human umbilical vein endothelial cells (HUVECs) allowed human cancer cells to transmigrate through a HUVEC monolayer. These results suggest that CLIC2 may be involved in the formation and/or maintenance of tight junctions and that cancer tissue vasculature lacks CLIC2 and tight junctions, which allows the intravasation of cancer cells necessary for hematogenous metastasis.

Significance of Glioma Stem-Like Cells in the Tumor Periphery That Express High Levels of CD44 in Tumor Invasion, Early Progression, and Poor Prognosis in Glioblastoma.

Glioblastoma multiforme (GBM) is the most aggressive malignant brain tumor and a subpopulation of glioma stem-like cells (GSCs) is likely responsible for the invariable recurrence following maximum resection and chemoradiotherapy. As most GSCs that are located in the perivascular and perinecrotic niches should be removed during tumor resection, it is very important to know where surviving GSCs are localized. Here, we investigated the existence and functions of GSCs in the tumor periphery, which is considered to constitute the invasion niche for GSCs in GBM, by analyzing expression of stem cell markers and stem cell-related molecules and measuring particular activities of cultured GSCs. In addition, the relationship between GSCs expressing particular stem cell markers and pathological features on MRI and prognosis in GBM patients was analyzed. We showed that GSCs that express high levels of CD44 are present in the tumor periphery. We also found that vascular endothelial growth factor (VEGF) is characteristically expressed at a high level in the tumor periphery. Cultured GSCs obtained from the tumor periphery were highly invasive and have enhanced migration phenotype, both of which were markedly inhibited by CD44 knockdown. Higher expression of CD44 in the tumor periphery than in the core was correlated with a highly invasive feature on MRI and was associated with early tumor progression and worse survival, whereas lower expression of CD44 in the tumor periphery corresponded to low invasion and was associated with longer survival. The low invasion type on MRI tended to show high levels of VEGF expression in the tumor periphery, thus presenting the tumor with high proliferative activity. These results imply the significance of GSCs with high levels of CD44 expression in the tumor periphery compared to the core, not only in tumor invasion but also rapid tumor progression and short survival in patients with GBM.