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.

Perioperative perampanel administration for early seizure prophylaxis in brain tumor patients.

Background: The efficacy of perioperative prophylactic antiepileptic drug therapy in "seizure-naïve" patients with brain tumor, including glioblastoma (GBM), remains controversial. This study investigated whether perampanel (PER) is effective and safe for preventing perioperative onset of epileptic seizures, so-called early seizure, in patients with brain tumors. Methods: Forty-five patients underwent tumor resection through craniotomy for a primary supratentorial brain tumor at Ehime University Hospital between April 2021 and July 2022. PER was administered from the 1st to the 6th day after surgery for seizure prophylaxis. Occurrence of early seizure, hematological toxicities, and various side effects were recorded on postoperative days 7 and 14. In addition, the clinical course of these patients was compared with 42 brain tumor patients under the same treatment protocol who received levetiracetam (LEV) for seizure prophylaxis between April 2017 and October 2018. Results: In 45 patients with brain tumor, including GBM, who received PER administration, no early seizures were identified within 7 days postoperatively. No adverse drug reactions such as hematological toxicity, liver or kidney dysfunction, or exanthematous drug eruption were observed in any cases. As side effects, somnolence was reported in 14 patients (31.1%), vertigo in 3 patients (6.7%), and headache in 3 patients (6.7%). Although somnolence and vertigo were difficult to assess in the case of intraparenchymal tumors, particularly GBM, these side effects were not identified in patients with extraparenchymal tumors such as meningiomas, epidermoid cysts, and pituitary adenomas. In addition, no significant differences were identified compared to patients who received LEV. Conclusion: The efficacy and safety of PER in preventing early seizures among patients with brain tumors were retrospectively evaluated. Perioperative administration of PER to patients with brain tumors may reduce the risk of early seizures without incurring serious side effects, showing no significant differences compared to patients who received LEV.

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.

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.

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.

Intracranial anaplastic solitary fibrous tumor/hemangiopericytoma: immunohistochemical markers for definitive diagnosis.

Intracranial anaplastic hemangiopericytoma (AHPC) is a rare and malignant subset of solitary fibrous tumor/hemangiopericytoma (SFT/HPC) as per the WHO 2016 Classification of Tumors of the Central Nervous System. AHPC portends a poor prognosis and is associated with higher rates of recurrence/metastasis in comparison with SFT/HPC. Accordingly, it is critical to continue to define the clinical course of patients with AHPC and in so doing further refine clinicopathologic/immunohistochemical (IHC) criteria needed for definitive diagnosis. Herein, we describe clinical/histological characteristics of six patients with AHPC. In addition, we reviewed and analyzed the expression of various IHC markers reported within the literature (i.e., a total of 354 intracranial SFT/HPCs and 460 meningiomas). Histologically, tumors from our six patients were characterized by a staghorn-like vascular pattern, mitotic cells, and strong nuclear atypia. Immunohistochemically, all tumors displayed positive nuclear staining for STAT6; other markers, including CD34 and Bcl-2, were expressed only in three patients. Analysis of IHC expression patterns for SFT/HPC and meningioma within the literature revealed that nuclear expression of STAT6 had the highest specificity (100%) for SFT/HPC, followed by ALDH1 (97.2%) and CD34 (93.6%). Of note, SSTR2A (95.2%) and EMA (85%) displayed a high specificity for meningioma. Anaplastic SFT/HPC is a tumor with poor prognosis that is associated with higher rates of recurrence and metastasis in comparison with SFT/HPC. Given that anaplastic SFT/HPC requires more aggressive treatment than meningioma despite of a similar presentation on imaging, it is crucial to be able to distinguish between these tumors.

Tumor edge-to-core transition promotes malignancy in primary-to-recurrent glioblastoma progression in a PLAGL1/CD109-mediated mechanism.

BACKGROUND: Glioblastoma remains highly lethal due to its inevitable recurrence. Most of this recurrence is found locally, indicating that postsurgical tumor-initiating cells (TICs) accumulate at the tumor edge. These edge-TICs then generate local recurrence harboring new core lesions. Here, we investigated the clinical significance of the edge-to-core (E-to-C) signature generating glioblastoma recurrence and sought to identify its central mediators. METHODS: First, we examined the association of E-to-C-related expression changes to patient outcome in matched primary and recurrent samples (n = 37). Specifically, we tested whether the combined decrease of the edge-TIC marker PROM1 (CD133) with the increase of the core-TIC marker CD109, representing E-to-C transition during the primary-to-recurrence progression, indicates poorer patient outcome. We then investigated the specific molecular mediators that trigger tumor recurrence driven by the E-to-C progression. Subsequently, the functional and translational significance of the identified molecule was validated with our patient-derived edge-TIC models in vitro and in vivo. RESULTS: Patients exhibiting the CD133low/CD109high signature upon recurrence representing E-to-C transition displayed a strong association with poorer progression-free survival and overall survival among all tested patients. Differential gene expression identified that PLAGL1 was tightly correlated with the core TIC marker CD109 and was linked to shorter patient survival. Experimentally, forced PLAGL1 overexpression enhanced, while its knockdown reduced, glioblastoma edge-derived tumor growth in vivo and subsequent mouse survival, suggesting its essential role in the E-to-C-mediated glioblastoma progression. CONCLUSIONS: E-to-C axis represents an ongoing lethal process in primary glioblastoma contributing to its recurrence, partly in a PLAGL1/CD109-mediated mechanism.

Usefulness of an Image Fusion Model Using Three-Dimensional CT and MRI with Indocyanine Green Fluorescence Endoscopy as a Multimodal Assistant System in Endoscopic Transsphenoidal Surgery.

Purpose. We investigate the usefulness of multimodal assistant systems using a fusion model of preoperative three-dimensional (3D) computed tomography (CT) and magnetic resonance imaging (MRI) along with endoscopy with indocyanine green (ICG) fluorescence in establishing endoscopic endonasal transsphenoidal surgery (ETSS) as a more effective treatment procedure. Methods. Thirty-five consecutive patients undergoing ETSS in our hospital between April 2014 and March 2015 were enrolled in the study. In all patients, fusion models of 3D-CT and MRI were created by reconstructing preoperative images. In addition, in 10 patients, 12.5 mg of ICG was intravenously administered, allowing visualization of surrounding structures. We evaluated the accuracy and utility of these combined modalities in ETSS. Results. The fusion model of 3D-CT and MRI clearly demonstrated the complicated structures in the sphenoidal sinus and the position of the internal carotid arteries (ICAs), even with extensive tumor infiltration. ICG endoscopy enabled us to visualize the surrounding structures by the phasic appearance of fluorescent signals emitted at specific consecutive elapsed times. Conclusions. Preoperative 3D-CT and MRI fusion models with intraoperative ICG endoscopy allowed distinct visualization of vital structures in cases where tumors had extensively infiltrated the sphenoidal sinus. Additionally, the ICG endoscope was a useful real-time monitoring tool for ETSS.

Two cases of pineal-region meningiomas derived from arachnoid membrane over the vein of Galen without dural attachment.

BACKGROUND: We present two rare cases of pineal-region meningiomas. These tumors are the first reported cases of dura-unrelated meningiomas originating from the arachnoid membrane over the vein of Galen (AMG). CASE DESCRIPTION: In Case 1, a 37-year-old woman presented with a progressing headache. Magnetic resonance imaging (MRI) showed a large tumor in the pineal region, displacing the vein of Galen upward. Angiography disclosed occlusion of the vein of Galen, with deep venous flow draining through the veins on the right medial surface of the occipital lobe to the superior sagittal sinus. In Case 2, a 67-year-old man presented with dizziness. MRI demonstrated a large mass in the pineal region, displacing the vein of Galen inferiorly. Angiography disclosed occlusion of the vein of Galen, with deep venous flow draining through the collateral venous channel into the transverse sinus. Both tumors were totally excised (Simpson Grade III for Case 1, Grade I for Case 2) via a left occipital transtentorial approach. No dural attachment was recognized in either case, but the tumor in Case 1 was firmly adherent to the inferior portion of the AMG, while that in Case 2 was attached to the superior portion of the AMG, but remained dissectible. CONCLUSIONS: We reported two cases of pineal-region meningiomas originating from the arachnoid membrane over the vein of Galen, resulting in meningioma without dural attachment. These tumors can be totally resected by careful dissection of the tumor from the arachnoid membrane surrounding the vein of Galen.

[A Case of Peripheral Middle Cerebral Artery Aneurysm Showing Repeated Morphological Changes].

We describe a case of a peripheral ruptured middle cerebral artery(MCA)aneurysm showing repeated morphological changes within a short period of 3 months. A 69-year-old woman was admitted to her primary care hospital with headache. Cranial computed tomography (CT) showed subarachnoid hemorrhage (SAH), but ruptured aneurysm was not confirmed on 4-vessel cerebral angiography. Conservative treatment was provided in the form of pain relief and blood pressure control. However, left internal carotid artery angiography (ICAG) conducted 12 days post-onset showed a peripheral MCA aneurysm, which was enlarged 1 week later. The patient did not tolerate balloon test occlusion, showing neurological deficit. Direct surgery was planned, but angiography on day 30 revealed a reduction in aneurysm size. Medical therapy was therefore continued for 1 month; however, the aneurysm showed repeated enlargements over 3 months. The patient therefore consulted our hospital for surgery, which was performed using a transsylvian approach. As we were unable to rule out pseudoaneurysm, we performed superficial temporal artery-MCA anastomosis as a form of trapping surgery. However, the lesion appeared to likely represent a congenital aneurysm when viewed macroscopically, so we performed neck clipping as a definitive treatment. Navigation and motor-evoked potential monitoring were useful to approach the aneurysm and predict the preservation of motor function. Histological examination revealed a congenital aneurysm with organized thrombus. The postoperative course was uneventful and the patient was discharged 2 weeks after surgery without any neurological deficit.

Usefulness of the fusion imaging of 3D-CT and MRA for carotid artery stenting with minimum use of contrast medium in patient with renal dysfunction.

Patients with severe internal carotid artery (ICA) stenosis with multiple medical problems generally undergo carotid artery stenting (CAS). However, it is difficult to perform CAS in some patients because iodinated contrast medium is hard to use. We report a patient with asymptomatic ICA stenosis and chronic renal failure, in whom successful treatment was achieved using CAS with minimal use of iodinated contrast medium. A 68-year-old man with severely chronic renal failure was consulted for treatment of left ICA stenosis. Magnetic resonance angiography (MRA) and carotid echography revealed left ICA severe stenosis, and systemic non-contrast MRA showed left femoral artery constriction, but right femoral artery to be intact. CAS was therefore performed through the right femoral artery, using non-contrast three-dimensional computed tomography (3D-CT) with MRA fusion imaging, intravascular ultrasonography, and a small amount of iodinated low-osmolar contrast medium. Postoperative course was uneventful with no aggravation of renal dysfunction, and he was discharged 7 days postoperatively. These techniques are very useful for patients with chronic renal failure, and this present case represents the first report of CAS treated by using non-contrast 3D-CT with MRA fusion image technique.