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.

Role of amide proton transfer imaging in maximizing tumor resection in malignant glioma: a possibility to take the place of 11C-methionine positron emission tomography.

BACKGROUND: Amide proton transfer (APT) imaging has been proposed as a technique to assess tumor metabolism. However, the relationship between APT imaging and other quantitative modalities including positron emission tomography (PET) has not been investigated in detail. This study aimed to evaluate the clinical usefulness of APT imaging in determining the metabolic status of malignant glioma and to compare findings with those from 11C-methionine (Met)-PET. METHODS: This research analyzed APT imaging data from 20 consecutive patients with malignant glioma treated between January 2022 and July 2023. Patients underwent tumor resection and correlations between tumor activity and intensity of APT signal were investigated. We also compared 11C-Met-PET and APT imaging for the same regions of the perifocal tumor invasion area. RESULTS: Clear, diagnostic APT images were obtained from all 20 cases. Mean APT intensity (APTmean) was significantly higher in the glioblastoma (GBM), IDH wild type group (27.2 ± 12.8%) than in other gliomas (6.0 ± 4.7%; p < 0.001). The cut-off APTmean to optimally distinguish between GBM and other malignant gliomas was 12.8%, offering 100% sensitivity and 83.3% specificity. These values for APTmean broadly matched the tumor-to-contralateral normal brain tissue ratio from 11C-Met-PET analysis (r = 0.66). The APT signal was also observed in the gadolinium non-contrast region on T1-weighted imaging, appearing to reflect the surrounding tumor-infiltrated area. CONCLUSIONS: APT imaging can be used to evaluate the area of tumor invasion, similar to 11C-Met-PET. APT imaging revealed low invasiveness in patients and was useful in preoperative planning for tumor resection, facilitating maximum tumor resection including the tumor invasive area.

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.

Rational Design, Multistep Synthesis and in Vitro Evaluation of Poly(glycerol) Functionalized Nanodiamond Conjugated with Boron-10 Cluster and Active Targeting Moiety for Boron Neutron Capture Therapy.

Boron neutron capture therapy (BNCT), advanced cancer treatment utilizing nuclear fission of ¹⁰ B atom in cancer cells, is attracting increasing attention. As ¹⁰ B delivery agent, sodium borocaptate (¹⁰ BSH, ¹⁰ B₁₂ H₁₁ SH ⋅ 2Na), has been used in clinical studies along with L-boronophenylalanine. Recently, this boron cluster has been conjugated with lipids, polymers or nanoparticles to increase selectivity to and retentivity in tumor. In this work, anticancer nanoformulations for BNCT are designed, consisting of poly(glycerol) functionalized detonation nanodiamonds (DND-PG) as a hydrophilic nanocarrier, the boron cluster moiety (¹⁰ B₁₂ H₁₁ ^2- ) as a dense boron-10 source, and phenylboronic acid or RGD peptide as an active targeting moiety. Some hydroxy groups in PG were oxidized to carboxy groups (DND-PG-COOH) to conjugate the active targeting moiety. Some hydroxy groups in DND-PG-COOH were then transformed to azide to conjugate ¹⁰ B₁₂ H₁₁ ^2- through click chemistry. The nanodrugs were evaluated in vitro using B16 murine melanoma cells in terms of cell viability, BNCT efficacy and cellular uptake. As a result, the ¹⁰ B₁₂ H₁₁ ^2- moiety is found to facilitate cellular uptake probably due to its negative charge. Upon thermal neutron irradiation, the nanodrugs with ¹⁰ B₁₂ H₁₁ ^2- moiety exhibited good anticancer efficacies with slight differences with and without targeting moiety.

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.

Rational Design, Multistep Synthesis and in Vitro Evaluation of Poly(glycerol) Functionalized Nanodiamond Conjugated with Boron-10 Cluster and Active Targeting Moiety for Boron Neutron Capture Therapy.

Boron neutron capture therapy (BNCT), advanced cancer treatment utilizing nuclear fission of 10 B atom in cancer cells, is attracting increasing attention. As 10 B delivery agent, sodium borocaptate (10 BSH, 10 B12 H11 SH ⋅ 2Na), has been used in clinical studies along with L-boronophenylalanine. Recently, this boron cluster has been conjugated with lipids, polymers or nanoparticles to increase selectivity to and retentivity in tumor. In this work, anticancer nanoformulations for BNCT are designed, consisting of poly(glycerol) functionalized detonation nanodiamonds (DND-PG) as a hydrophilic nanocarrier, the boron cluster moiety (10 B12 H11 2- ) as a dense boron-10 source, and phenylboronic acid or RGD peptide as an active targeting moiety. Some hydroxy groups in PG were oxidized to carboxy groups (DND-PG-COOH) to conjugate the active targeting moiety. Some hydroxy groups in DND-PG-COOH were then transformed to azide to conjugate 10 B12 H11 2- through click chemistry. The nanodrugs were evaluated in vitro using B16 murine melanoma cells in terms of cell viability, BNCT efficacy and cellular uptake. As a result, the 10 B12 H11 2- moiety is found to facilitate cellular uptake probably due to its negative charge. Upon thermal neutron irradiation, the nanodrugs with 10 B12 H11 2- moiety exhibited good anticancer efficacies with slight differences with and without targeting moiety.

Complex Dispersion of Detonation Nanodiamond Revealed by Machine Learning Assisted Cryo-TEM and Coarse-Grained Molecular Dynamics Simulations.

Understanding the polydispersity of nanoparticles is crucial for establishing the efficacy and safety of their role as drug delivery carriers in biomedical applications. Detonation nanodiamonds (DNDs), 3-5 nm diamond nanoparticles synthesized through detonation process, have attracted great interest for drug delivery due to their colloidal stability in water and their biocompatibility. More recent studies have challenged the consensus that DNDs are monodispersed after their fabrication, with their aggregate formation poorly understood. Here, we present a novel characterization method of combining machine learning with direct cryo-transmission electron microscopy imaging to characterize the unique colloidal behavior of DNDs. Together with small-angle X-ray scattering and mesoscale simulations we show and explain the clear differences in the aggregation behavior between positively and negatively charged DNDs. Our new method can be applied to other complex particle systems, which builds essential knowledge for the safe implementation of nanoparticles in drug delivery.

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.

Steric Interaction of Polyglycerol-Functionalized Detonation Nanodiamonds.

Detonation nanodiamonds have found numerous potential applications in a diverse array of fields such as biomedical imaging and drug delivery. Here, we systematically characterized non-functionalized and polyglycerol-functionalized detonation nanodiamond particles (DNPs) dispersed in aqueous suspensions at different ionic strengths (∼1.0 × 10-7 to 1.0 × 10-2 M) via dynamic light scattering and cryogenic transmission electron microscopy. For these colloidal suspensions, the total potential energies of interactions between a pair of DNPs were theoretically calculated using the Derjaguin-Landau-Verwey-Overbeek (DLVO) theory plus the fitting of the Boltzmann distribution to the interparticle spacing distribution of the colloidal DNPs. These investigations revealed that the non-functionalized DNPs are dispersed in aqueous media through the long-range (>10 nm) and weak (<7 kBT) electrical double-layer repulsive interaction, while the driving force on dispersion of polyglycerol-functionalized DNPs is mostly derived from the short-range (<2 nm) and strong (∼55 kBT) steric repulsive potential barrier generated by the polyglycerol. Moreover, our results show that the truly monodispersed and individually dispersed DNP colloids, forming no aggregates in aqueous suspensions, are available by both functionalizing DNPs by polyglycerol and increasing ionic strength of suspending media to ≳1.0 × 10-2 M.

[Examination of Factors Related to Walking Ability in Patients with Abdominal Cancer].

It has been reported that preoperative rehabilitation reduces the risk of postoperative complications. We examined the factors impacting the efficacy of preoperative rehabilitation. Forty-three cancer patients who underwent abdominal surgery after preoperative rehabilitation at our hospital were assessed". Walkable"was defined as having the ability to walk to the toilet(distance>30 meters)without requiring support. Following the clinical path, if patients became"walkable"by the second day after surgery, they belonged to the smooth group, while the remaining patients belonged to the delayed group. We examined the factors influencing walking ability. The smooth group consisted of 34 patients(79%), and the delayed group consisted of 9 patients(21%). The significant factors related to delays in acquiring walking ability were old age and weakened lower limb function. Improving lower limb function through preoperative rehabilitation may lead to patients acquiring walking ability earlier after surgery, especially in older patients.

Utility of targeted balloon protection of the venous sinus for endovascular treatment of dural arteriovenous fistula by transarterial embolization with Onyx: A case report and literature review.

BACKGROUND: Onyx has already been reported as an effective and safe agent in transarterial embolization of cranial dural arteriovenous fistula (d-AVF). However, successful treatment is related to not only complete shunt obliteration but also preservation of a normal route of venous drainage. Here, we present a case of transverse sigmoid d-AVF in which successful treatment was achieved by transarterial Onyx embolization with targeted balloon protection of the venous drainage. CASE DESCRIPTION: A 70-year-old man presented with a 3-month history of tinnitus in the left ear and mild headache. Magnetic resonance imaging (MRI) showed a cluster of abnormal blood vessels in the area of the left transverse sinus (TS)-sigmoid sinus (SS) junction. Cerebral angiography demonstrated a Cognard type IIa d-AVF at the left TS-SS junction, supplied mainly by vessels such as the left middle meningeal artery, left occipital artery, and left meningohypophyseal trunk. In the venous phase, the ipsilateral TS-SS was recognized as a functional sinus and the left vein of Labbe drained into the TS near the drainage channel. Based on these findings, we decided to perform endovascular treatment under a transarterial approach with Onyx using targeted balloon protection of the venous sinus to protect against Onyx migration and preserve antegrade sinus flow. The patient recovered well without sequelae, and follow-up MRI 12 months later showed complete disappearance of the d-AVF. CONCLUSION: This treatment strategy using targeted balloon protection may be very useful to preserve antegrade sinus flow in patients with Cognard type IIa d-AVF.

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.

Effect of Protein Corona on Mitochondrial Targeting Ability and Cytotoxicity of Triphenylphosphonium Conjugated with Polyglycerol-Functionalized Nanodiamond.

Functionalization of nanoparticles (NPs) with targeting moieties has a high potential to advance precision nanomedicine. However, the targeting moieties on a NP surface are known to be masked by a protein corona in biofluids, lowering the targeting efficiency. Although it has been demonstrated at the cellular level, little is known about the influence of the protein corona on the subcellular targeting. Herein, we adopted triphenylphosphonium (TPP) as a mitochondrial targeting moiety and investigated the effects of protein coronas from fetal bovine serum and human plasma on its targeting ability and cytotoxicity. Specifically, we introduced TPP in low (l) and high (h) densities on the surface of nanodiamond (ND) functionalized with polyglycerol (PG). Despite the "corona-free" PG interface, we found that the TPP moiety attracted proteins to form a corona layer with clear linearity between the TPP density and the protein amount. By performing investigations on human cervix epithelium (HeLa) and human lung epithelial carcinoma (A549) cells, we further demonstrated that (1) the protein corona alleviated the cytotoxicity of both ND-PG-TPP-l and -h, (2) a smaller amount of proteins on the surface of ND-PG-TPP-l did not affect its mitochondrial targeting ability, and (3) a larger amount of proteins on the surface of ND-PG-TPP-h diminished its targeting specificity by restricting the NDs inside the endosome and lysosome compartments. Our findings will provide in-depth insights into the design of NPs with active targeting moiety for more precise and safer delivery at the subcellular level.

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.

Met-PET uptake index for total tumor resection: identification of 11C-methionine uptake index as a goal for total tumor resection including infiltrating tumor cells in glioblastoma.

Glioblastoma multiforme (GBM) is largely due to glioma stem cells (GSCs) that escape from total resection of gadolinium (Gd)-enhanced tumor on MRI. The aim of this study is to identify the imaging requirements for maximum resection of GBM with infiltrating GSCs. We investigated the relationship of tumor imaging volume between MRI and 11C-methionine (Met)-PET and also the relationship between Met uptake index and tumor activity. In ten patients, tumor-to-contralateral normal brain tissue ratio (TNR) was calculated to evaluate metabolic activity of Met uptake areas which were divided into five subareas by the degrees of TNR. In each GBM, tumor tissue was obtained from subareas showing the positive Met uptake. Immunohistochemistry was performed to examine the tumor proliferative activity and existence of GSCs. In all patients, the volume of Met uptake area at TNR ≦ 1.4 was larger than that of the Gd-enhanced area. The Met uptake area at TNR 1.4 beyond the Gd-enhanced tumor was much wider in high invasiveness-type GBMs than in those of low invasiveness type, and survival was much shorter in the former than the latter types. Immunohistochemistry revealed the existence of GSCs in the area showing Met uptake at TNR 1.4 and no Gd enhancement. Areas at TNR > 1.4 included active tumor cells with relatively high Ki-67 labeling index. In addition, it was demonstrated that GSCs could exist beyond the border of Gd-enhanced tumor. Therefore, to obtain maximum resection of GBMs, including infiltrating GSCs, aggressive surgical excision that includes the Met-positive area at TNR 1.4 should be considered.

Tricks and traps of ICG endoscopy for effectively applying endoscopic transsphenoidal surgery to pituitary adenoma.

Differentiating tumor from normal pituitary gland is very important for achieving complete resection without complications in endoscopic endonasal transsphenoidal surgery (ETSS) for pituitary adenoma. To facilitate such surgery, we investigated the utility of indocyanine green (ICG) fluorescence endoscopy as a tool in ETSS. Twenty-four patients with pituitary adenoma were enrolled in the study and underwent ETSS using ICG endoscopy. After administering 12.5 mg of ICG twice an operation with an interval > 30 min, times from ICG administration to appearance of fluorescence on vital structures besides the tumor were measured. ICG endoscopy identified vital structures by the phasic appearance of fluorescent signals emitted at specific consecutive elapsed times. Elapsed times for internal carotid arteries did not differ according to tumor size. Conversely, as tumor size increased, elapsed times for normal pituitary gland were prolonged but those for the tumor were reduced. ICG endoscopy revealed a clear boundary between tumors and normal pituitary gland and enabled confirmation of no more tumor. ICG endoscopy could provide a useful tool for differentiating tumor from normal pituitary gland by evaluating elapsed times to fluorescence in each structure. This method enabled identification of the boundary between tumor and normal pituitary gland under conditions of a low-fluorescence background, resulting in complete tumor resection with ETSS. ICG endoscopy will contribute to improve the resection rate while preserving endocrinological functions in ETSS for pituitary adenoma.