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PDF file, 60KB, Clinical characteristics of GBM tissue microarray.

PDF file, 84KB, Antibodies used for western analysis and subclassification of human glioblastoma stem-like cells.

PDF file, 78KB, Antibodies used for immunohistochemical analysis of human glioblastoma stem-like cell induced mouse xenograft tumors and grade IV astrocytoma tissue microarray.

PDF file, 80KB, Differential neural lineage marker expression associate with survivability of GSC-induced xenografts in immunodeficient mice.

Purpose: Glioblastoma multiforme (GBM) is a poorly treated human brain cancer with few established clinically useful molecular prognostic markers. We characterized glioblastoma stem–like cells (GSC) according to developmental neural lineage markers and correlated their expression with patient survival.Experimental Design: Immunoblot array of neural lineage markers classified five independently isolated human GSC lines into three classes exhibiting differential expression of oligodendrocyte progenitor cells (OPC), astrocyte progenitor cells (APC), and neural progenitor cells (NPC) markers. Immunodeficient mice were orthotopically implanted with each cell line to evaluate tumor infiltration and recipient survival. 2′,3′-Cyclic-nucleotide 3′-phosphodiesterase (CNP) antigenic expression was used to evaluate a clinically annotated GBM tissue microarray with 115 specimens.Results: We report that molecular classification of patient-derived GSCs using neural lineage markers show association with differential xenograft invasiveness, and also show significant correlation to survival in both the mouse model and human patients. Orthotopic implantation into immunodeficient mice showed Ki-67 proliferative index independent xenograft infiltration: class I GSCs (OPC and NPC positive) established focal lesions, class II GSCs (NPC positive) formed minimally invasive lesions, and class III GSCs (APC positive) established highly infiltrative lesions. The OPC marker, CNP also exhibited high expression in focal xenografts versus low expression in invasive xenografts. Differential CNP expression correlated with mouse model survival, and CNP immunoassay of a large GBM tissue microarray also showed significant differential patient survival.Conclusions: GSC classification with developmental neural lineage markers revealed CNP as a novel and potentially useful clinical prognosis marker, and suggests clinical importance for patient-specific GSC analysis. Clin Cancer Res; 18(13); 3628–36. ©2012 AACR.

PDF file, 327KB, Low and high magnification histology and IHC of differential tumor infiltration in mouse xenografts.

Purpose: Glioblastoma is the most malignant primary brain tumor, with a median survival of less than 2 years. More effective therapeutic approaches are needed to improve clinical outcomes.Experimental Design: Glioblastoma patient-derived cells (GPDC) were isolated from patient glioblastomas and implanted in mice to form xenografts. IHC was performed for human Ether-à-go-go-Related Gene (hERG) expression and tumor proliferation. Sphere-forming assays with the hERG blocker E-4031 were performed on a high and low hERG–expressing lines. A glioblastoma tissue microarray (TMA; 115 patients) was used to correlate hERG expression with patient survival. Clinical data were analyzed to determine whether patient survival was affected by incidental administration of hERG inhibitory drugs and the correlative effect of patient glioblastoma hERG expression levels.Results: hERG expression was upregulated in glioblastoma xenografts with higher proliferative indices. High hERG–expressing GPDCs showed a reduction in sphere formation when treated with hERG inhibitors compared with low hERG–expressing GPDCs. Glioblastoma TMA analysis showed worse survival for glioblastoma patients with high hERG expression versus low expression—43.5 weeks versus 60.9 weeks, respectively (P = 0.022). Furthermore, patients who received at least one hERG blocker had a better survival rate compared with patients who did not (P = 0.0015). Subgroup analysis showed that glioblastoma patients with high hERG expression who received hERG blockers had improved survival (P = 0.0458). There was no difference in survival for low hERG–expressing glioblastoma patients who received hERG blockers (P = 0.4136).Conclusions: Our findings suggest that hERG is a potential glioblastoma survival marker, and that already approved drugs with non-torsadogenic hERG inhibitory activity may potentially be repurposed as adjuvant glioblastoma therapy in high hERG–expressing glioblastoma patients. Clin Cancer Res; 23(1); 73–80. ©2016 AACR.See related commentary by Arcangeli and Becchetti, p. 3

The number of patients who received each combination of hERG blockers is shown.

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Malignant brain tumors are among the most intractable cancers, including malignancies such as glioblastoma, diffuse midline glioma, medulloblastoma, and ependymoma. Unfortunately, treatment options for these brain tumors have been inadequate and complex, leading to poor prognoses and creating a need for new treatment modalities. Aberrant epigenetics define these types of tumors, with underlying changes in DNA methylation, histone modifications, chromatin structure and noncoding RNAs. Epigenetic-targeted therapies are an alternative that have the potential to reverse the epigenetic deregulation underpinning brain malignancies. Various drugs targeting epigenetic regulators have shown promise in preclinical and clinical testing. In this review, we highlight some of the recent emerging epigenetic targeted therapies for brain tumors being evaluated in the discovery phase and in clinical trials.

Extracellular matrix (ECM) is a rich mixture of proteins and glycans secreted by cells. This includes typical ECM structures such as collagen and heparin as well as glycosylated, secreted proteins such as growth factors and peptidases. Certain components of ECM are ubiquitous among all tissue; however, each biological tissue also displays unique variations that can be identified using biopanning techniques. Here we describe using a variable lymphocyte receptor (VLR) yeast surface display library to identify selective binders to brain ECM by combining ECM biopanning with a rapid ELISA-based screen using clonal VLRs isolated directly from the yeast surface. Finally, potential ECM-binding candidates can be verified by immunostaining murine brain sections with VLRs released from the yeast surface. These methods provide a framework for the identification of tissue-selective ECM-binding VLRs using yeast surface display techniques and could easily be adapted for other binding scaffolds or ECM from other tissues.

Functional resonance magnetic imaging (fMRI) allows for identification of eloquent cortex in pre-treatment planning. Previous studies have shown a correlation among lesion to activation distance (LAD) measures and morbidity and mortality. This study investigates the relationship between LAD, well-established language centers (Wernicke’s and Broca’s), and language performance measures. We included a sample population of brain tumor patients that received language fMRI (verbal fluency and sentence verification) for pre-treatment assessment (n = 51). LAD to the nearest language area was measured and divided into groups ≤ 10 mm and > 10 mm. Verbal fluency scores were compared between these groups. Additionally, patients were divided into similar groups based on LAD to either Broca’s or Wernicke’s areas, and the verbal fluency scores and sentence verification accuracy (n = 29) were subsequently compared between groups. Brain tumor patients with LAD ≤ 10 mm to either language area had significantly lower verbal fluency scores (p = 0.028). The difference in verbal fluency scores between groups with LAD ≤ 10 mm and > 10 mm to Wernicke’s area trends toward significance (p = 0.067). The sentence verification accuracy was significantly lower in patients with LAD ≤ 10 mm to either language area (p = 0.039). These findings suggest that there exists a significant relationship between LAD to language centers and measures; greater language deficits are seen when LAD ≤ 10 mm.

Alkylphosphocholine (APC) analogs are a novel class of broad-spectrum tumor-targeting agents that can be used for both diagnosis and treatment of cancer. The potential for clinical translation for APC analogs will strongly depend on their pharmacokinetic (PK) profiles. The aim of this work was to understand how the chemical structures of various APC analogs impact binding and PK. To achieve this aim, we performed in silico docking analysis, in vitro and in vivo partitioning experiments, and in vivo PK studies. Our results have identified 7 potential high-affinity binding sites of these compounds on human serum albumin (HSA) and suggest that the size of the functional group directly influences the albumin binding, partitioning, and PK. Namely, the bulkier the functional groups, the weaker the agent binds to albumin, the more the agent partitions onto lipoproteins, and the less time the agent spends in circulation. The results of these experiments provide novel molecular insights into the binding, partitioning, and PK of this class of compounds and similar molecules, as well as suggests pharmacological strategies to alter their PK profiles. Importantly, our methodology may provide a way to design better drugs by better characterizing PK profile for lead compound optimization.

Please see the full-text version of this guideline https://www.cns.org/guidelines/guidelines-treatment-adults-metastatic-brain-tumors/chapter_2) for the target population of each recommendation listed below. SURGERY FOR METASTATIC BRAIN TUMORS AT NEW DIAGNOSIS QUESTION: Should patients with newly diagnosed metastatic brain tumors undergo surgery, stereotactic radiosurgery (SRS), or whole brain radiotherapy (WBRT)? Recommendations Level 1: Surgery + WBRT is recommended as first-line treatment in patients with single brain metastases with favorable performance status and limited extracranial disease to extend overall survival, median survival, and local control. Level 3: Surgery plus SRS is recommended to provide survival benefit in patients with metastatic brain tumors Level 3: Multimodal treatments including either surgery + WBRT + SRS boost or surgery + WBRT are recommended as alternatives to WBRT + SRS in terms of providing overall survival and local control benefits. SURGERY AND RADIATION FOR METASTATIC BRAIN TUMORS QUESTION: Should patients with newly diagnosed metastatic brain tumors undergo surgical resection followed by WBRT, SRS, or another combination of these modalities? Recommendations Level 1: Surgery + WBRT is recommended as superior treatment to WBRT alone in patients with single brain metastases. Level 3: Surgery + SRS is recommended as an alternative to treatment with SRS alone to benefit overall survival. Level 3: It is recommended that SRS alone be considered equivalent to surgery + WBRT. SURGERY FOR RECURRENT METASTATIC BRAIN TUMORS QUESTION: Should patients with recurrent metastatic brain tumors undergo surgical resection? Recommendations Level 3: Craniotomy is recommended as a treatment for intracranial recurrence after initial surgery or SRS. SURGICAL TECHNIQUE AND RECURRENCE QUESTION A: Does the surgical technique (en bloc resection or piecemeal resection) affect recurrence? Recommendation Level 3: En bloc tumor resection, as opposed to piecemeal resection, is recommended to decrease the risk of postoperative leptomeningeal disease when resecting single brain metastases. Question b Does the extent of surgical resection (gross total resection or subtotal resection) affect recurrence? Recommendation Level 3: Gross total resection is recommended over subtotal resection in recursive partitioning analysis class I patients to improve overall survival and prolong time to recurrence. The full guideline can be found at https://www.cns.org/guidelines/guidelines-treatment-adults-metastatic-brain-tumors/chapter_2.

Focused ultrasound transiently opens the blood-brain barrier and provides the potential to modulate tumor immunostimulatory effect., Focused ultrasound (FUS) in the presence of microbubbles can transiently open the blood-brain barrier (BBB) to increase therapeutic agent penetration at the targeted brain site to benefit recurrent glioblastoma (rGBM) treatment. This study is a dose-escalating pilot trial using a device combining neuronavigation and a manually operated frameless FUS system to treat rGBM patients. The safety and feasibility were established, while a dose-dependent BBB-opening effect was observed, which reverted to baseline within 24 hours after treatment. No immunological response was observed clinically under the applied FUS level in humans; however, selecting a higher level in animals resulted in prolonged immunostimulation, as confirmed preclinically by the recruitment of lymphocytes into the tumor microenvironment (TME) in a rat glioma model. Our findings provide preliminary evidence of FUS-induced immune modulation as an additional therapeutic benefit by converting the immunosuppressive TME into an immunostimulatory TME via a higher but safe FUS dosage.

Recent research has highlighted the importance of key tumor microenvironment features, notably the collagen-rich extracellular matrix (ECM) in characterizing tumor invasion and progression. This led to great interest from both basic researchers and clinicians, including pathologists, to include collagen fiber evaluation as part of the investigation of cancer development and progression. Fibrillar collagen is the most abundant in the normal extracellular matrix, and was revealed to be upregulated in many cancers. Recent studies suggested an emerging theme across multiple cancer types in which specific collagen fiber organization patterns differ between benign and malignant tissue and also appear to be associated with disease stage, prognosis, treatment response, and other clinical features. There is great potential for developing image-based collagen fiber biomarkers for clinical applications, but its adoption in standard clinical practice is dependent on further translational and clinical evaluations. Here, we offer a comprehensive review of the current literature of fibrillar collagen structure and organization as a candidate cancer biomarker, and new perspectives on the challenges and next steps for researchers and clinicians seeking to exploit this information in biomedical research and clinical workflows.

BACKGROUND Radiomics and connectome analysis are distinct and non-invasive methods of deriving biologic information from MRI. Radiomics analyzes features intrinsic to the tumor, and connectomics incorporates data regarding the tumor and surrounding neural circuitry. In this study we used both techniques to predict glioma survival. METHODS We retrospectively identified 305 adult patients with histopathologically confirmed WHO grade II–IV gliomas who had presurgical, 3D, T1-weighted brain MRI. Available clinical variables included tumor lobe, hemisphere, multifocal nature grade, histology extent of surgical resection, patient age gender. For connectomics, we calculated nodal efficiencies, network size and degree for all pairs of 33 voxel cubes spanning the entire gray matter volume using similarity-based extraction and graph theory. Radiomic features were extracted using Pyradiomics and subjected to patient-level and population-level clustering (N=172). These clusters were then used to construct a multi-regional spatial interaction matrix for model building. Cox proportional hazards models were fit for clinical variables alone, connectomics alone, radiomics alone, connectomics+clinical and radiomics+clinical. We implemented 10-folds cross-validation and examined the mean area under the curve (AUC) across validation loops. RESULTS Median survival time was 134.2 months. The mean AUC for the clinical model was 0.79 +/- 0.01, the connectome model was 0.88 +/- 0.01, the combined connectome + clinical model was 0.93 +/- 0.01, the radiomic model was 0.64 +/- 0.05 and the radiomics+clinical model was 0.89+/-0.03. Radiomic analysis of the entire dataset as well as comparisons of radiomic+connectomics +/- clinical models are pending. CONCLUSIONS The combination of clinical variables and connectome analysis provided a more robust predictive model than other models. This suggests that connectome analysis incorporates valuable clinically-predictive information which can augment our capacity for prognostication of patients with diffuse glioma. These methods warrant further evaluation in larger prospective study of patients with diffuse glioma.

The brain vasculature maintains brain homeostasis by tightly regulating ionic, molecular, and cellular transport between the blood and the brain parenchyma. These blood–brain barrier (BBB) properties are impediments to brain drug delivery, and brain vascular dysfunction accompanies many neurological disorders. The molecular constituents of brain microvascular endothelial cells (BMECs) and pericytes, which share a basement membrane and comprise the microvessel structure, remain incompletely characterized, particularly in humans. To improve the molecular database of these cell types, we performed RNA sequencing on brain microvessel preparations isolated from snap-frozen human and mouse tissues by laser capture microdissection (LCM). The resulting transcriptome datasets from LCM microvessels were enriched in known brain endothelial and pericyte markers, and global comparison identified previously unknown microvessel-enriched genes. We used these datasets to identify mouse-human species differences in microvessel-associated gene expression that may have relevance to BBB regulation and drug delivery. Further, by comparison of human LCM microvessel data with existing human BMEC transcriptomic datasets, we identified novel putative markers of human brain pericytes. Together, these data improve the molecular definition of BMECs and brain pericytes, and are a resource for rational development of new brain-penetrant therapeutics and for advancing understanding of brain vascular function and dysfunction.

BackgroundImmune checkpoint inhibition (ICI) alone is not efficacious for a large number of patients with melanoma brain metastases. We previously established an in situ vaccination (ISV) regimen combining radiation and immunocytokine to enhance response to ICIs. Here, we tested whether ISV inhibits the development of brain metastases in a murine melanoma model.MethodsB78 (GD2+) melanoma ‘primary’ tumors were engrafted on the right flank of C57BL/6 mice. After 3–4 weeks, primary tumors were treated with ISV (radiation (12 Gy, day 1), α-GD2 immunocytokine (hu14.18-IL2, days 6–10)) and ICI (α-CTLA-4, days 3, 6, 9). Complete response (CR) was defined as no residual tumor observed at treatment day 90. Mice with CR were tested for immune memory by re-engraftment with B78 in the left flank and then the brain. To test ISV efficacy against metastases, tumors were also engrafted in the left flank and brain of previously untreated mice. Tumors were analyzed by quantitative reverse transcription-PCR, immunohistochemistry, flow cytometry and multiplex cytokine assay.ResultsISV+α-CTLA-4 resulted in immune memory and rejection of B78 engraftment in the brain in 11 of 12 mice. When B78 was engrafted in brain prior to treatment, ISV+α-CTLA-4 increased survival compared with ICI alone. ISV+α-CTLA-4 eradicated left flank tumors but did not elicit CR at brain sites when tumor cells were engrafted in brain prior to ISV. ISV+α-CTLA-4 increased CD8+ and CD4+ T cells in flank and brain tumors compared with untreated mice. Among ISV + α-CTLA-4 treated mice, left flank tumors showed increased CD8+ infiltration and CD8+:FOXP3+ ratio compared with brain tumors. Flank and brain tumors showed minimal differences in expression of immune checkpoint receptors/ligands or Mhc-1. Cytokine productions were similar in left flank and brain tumors in untreated mice. Following ISV+α-CTLA-4, production of immune-stimulatory cytokines was greater in left flank compared with brain tumor grafts.ConclusionISV augmented response to ICIs in murine melanoma at brain and extracranial tumor sites. Although baseline tumor-immune microenvironments were similar at brain and extracranial tumor sites, response to ISV+α-CTLA-4 was divergent with reduced infiltration and activation of immune cells in brain tumors. Additional therapies may be needed for effective antitumor immune response against melanoma brain metastases.

PURPOSE: There is a continuous search for imaging techniques with high sensitivity and specificity for brain tumors. Positron emission tomography (PET) imaging has shown promise, though many PET agents either have a low tumor specificity or impractical physical half-lives. [(124)I]CLR1404 is a small molecule alkylphosphocholine analogue that is thought to bind to plasma membrane lipid rafts and has shown high tumor-to-background ratios (TBR) in a previous pilot study in brain tumor patients. This study attempts to define the clinical value of [(124)I]CLR1404 PET/CT (aka CLR124). PROCEDURES: Adult patients with new or suspected recurrence of high grade primary or metastatic brain tumors (N = 27) were injected with [(124)I]CLR1404 followed by PET/CT at 6-, 24-, and 48-h. Standard uptake values (SUV) and TBR values were calculated for all time points. Uptake of [(124)I]CLR1404 was qualitatively assessed, compared with magnetic resonance imaging (MRI), and correlated with clinical outcome. Final diagnosis (N = 25) was established based on surgically resected tissue or long-term follow-up. RESULTS: Positive uptake with high TBR was detected in all but one patient with a final diagnosis of primary/recurrent brain tumor (12/13), and in less than half of patients with treatment-related changes (5/12). Concordance between [(124)I]CLR1404 uptake and contrast enhancement on MRI was seen in

Significance: Glioblastoma multiforme (GBM) is the most frequently diagnosed adult primary brain malignancy with poor patient prognosis. GBM can recur despite aggressive treatment due to therapeutically resistant glioblastoma stem cells (GSCs) that may exhibit metabolic plasticity. Aim: Intrinsic nicotinamide adenine dinucleotide (NADH) fluorescence can be acquired with fluorescence lifetime imaging microscopy (FLIM) to examine its bound and free metabolic states in GSC and GBM tissues. Approach: We compared the mean NADH fluorescence lifetime in live human GSCs and normal neural stem cells and validated those results by measuring oxygen consumption rates (OCRs). We also examined the role that invasive versus less-invasive GSCs had on tumor metabolism by measuring the mean NADH lifetimes and the relative amount of the longer-lived component of NADH and correlated these results with survival in an orthotopic mouse xenograft model. Results: Mean NADH lifetime, amount of bound NADH, and OCR were increased in GSCs. Compared with normal mouse brain, mean NADH lifetimes were longer for all GBM tissues. Invasive xenografts had higher relative amounts of the longer-lived NADH component, and this correlated with decreased survival. Conclusions: FLIM offers cellular resolution quantification of metabolic flux in GBM phenotypes, potentially informing biomedical researchers on improved therapeutic approaches.

// Hisham S. Alhajala 1 , John L. Markley 2 , Jin Hae Kim 2 , Mona M. Al-Gizawiy 3 , Kathleen M. Schmainda 3 , John S. Kuo 4 and Christopher R. Chitambar 1 , 3 1 Division of Hematology and Oncology, Department of Medicine, Medical College of Wisconsin, Milwaukee, Wisconsin, USA 2 Department of Biochemistry, University of Wisconsin-Madison, Madison, Wisconsin, USA 3 Department of Biophysics, Medical College of Wisconsin, Milwaukee, Wisconsin, USA 4 Department of Neurosurgery and Mulva Clinic for the Neurosciences, Dell Medical School, Austin, Texas, USA Correspondence to: Christopher R. Chitambar, email: chitambr@mcw.edu Keywords: glioblastoma; iron; gallium; metformin; mitochondrial complex I Received: February 01, 2020 Accepted: April 03, 2020 Published: April 28, 2020 ABSTRACT New drugs are needed for glioblastoma, an aggressive brain tumor with a dismal prognosis. We recently reported that gallium maltolate (GaM) retards the growth of glioblastoma in a rat orthotopic brain tumor model by inhibiting mitochondrial function and iron-dependent ribonucleotide reductase (RR). However, GaM’s mechanism of action at the mitochondrial level is not known. Given the interaction between gallium and iron metabolism, we hypothesized that gallium might target iron-sulfur (Fe-S) cluster-containing mitochondrial proteins. Using Extracellular Flux Analyzer technology, we confirmed that after a 24-h incubation, GaM 50 μmol/L inhibited glioblastoma cell growth by

Glioblastoma (GB) is highly invasive and resistant to multimodal treatment partly due to distorted vasculature and exacerbated inflammation. The aggressiveness of brain tumors may be attributed to the dysregulated release of angiogenic and inflammatory factors. The glycoprotein pentraxin-3 (PTX3) is correlated with the severity of some cancers. However, the mechanism responsible for the invasive oncogenic role of PTX3 in GB malignancy remains unclear. In this study, we examined the role of PTX3 in GB growth, angiogenesis, and invasion using in vitro and in vivo GB models, proteomic profiling, molecular and biochemical approaches. Under in vitro conditions, PTX3 over-expression in U87 cells correlated with cell cycle progression, increased migratory potential, and proliferation under hypoxic conditions. Conditioned media containing PTX3 enhanced the angiogenic potential of endothelial cells. While silencing of PTX3 by siRNA decreased the proliferation, migration, and angiogenic potential of U87 cells in vitro. Importantly, PTX3 over-expression increased tumor growth, angiogenesis, and invasion in an orthotopic mouse model. Higher levels of PTX3 in these tumors were associated with the upregulation of inflammatory and angiogenic markers including interleukin-8 (IL-8) and vascular endothelial growth factor (VEGF), but decreased levels of thrombospondin-1, an anti-angiogenic factor. Mechanistically, exogenous production of PTX3 triggered an IKK/NFκB signaling pathway that enhances the expression of the motility genes AHGEF7 and Rac1. Taken together, PTX3 expression is dysregulated in GB. PTX3 may augment invasion through enhanced angiogenesis in the GB microenvironment through the IL8-VEGF axis. Thus, PTX3 may represent a potential therapeutic target to mitigate the aggressive behavior of gliomas.

We report our initial experience using Minimally-Invasive Surgery (MIS) technique for Posterior Fossa Decompression (PFD) in Adult Chiari 1 Malformation (C1M) patients. Five subjects who were treated with MIS PFD at our center and followed up over a 5-year period. Another nine subjects who were treated with Open PFD and follow up over the same period were used for comparison. This study suggests that there are little differences in efficacy and safety between MIS and Open PFD. Larger series and prospective randomized trials comparing the two methods would provide higher-quality evidence and clarify the role of either technique in the treatment of C1M.

Background: Functional Magnetic Resonance Imaging (fMRI) is a presurgical planning technique used to localize functional cortex so as to maximize resection of diseased tissue and avoid viable tissue. In this retrospective study, we examined differences in morbidity and mortality of brain tumor patients who received preoperative fMRI in comparison to those who did not. Methods: Brain tumor patients (n = 206) were selected from a retrospective review of neurosurgical case logs from 2001 to 2009 at the University of Wisconsin-Madison. Results: Univariate analysis showed improved mortality in the fMRI group and the fMRI + Electrical Cortical Stimulation Mapping (ECM) group compared to the No-fMRI group. Multivariate analyses showed improved mortality of the fMRI group and the fMRI + ECM group compared to the No-fMRI group, with age and tumor grade being the most significant influencers. Overall, the fMRI group showed survival benefits at 3 years; twice that of the No-fMRI group. Furthermore, patients with high-grade tumors showed significant survival benefits in the fMRI group, while patients with low-grade tumors did not (controlling for age and ECM). There was also a significant difference in the two groups with respect to morbidity, with patients receiving fMRI showing improved outcomes in the motor and language domains. Conclusions: This study analyzing a large retrospective series of brain tumor patients with and without the use of fMRI in the preoperative planning has resulted in improved mortality and morbidity outcomes with the use of fMRI. These results point to the importance of incorporating fMRI in presurgical planning in the clinical management of patients with brain tumors. Keywords: Brain mapping, Brain tumors, Functional neuroimaging

Gallium, a metal with antineoplastic activity, binds transferrin (Tf) and enters tumor cells via Tf receptor1 (TfR1); it disrupts iron homeostasis leading to cell death. We hypothesized that TfR1 on brain microvascular endothelial cells (BMEC) would facilitate Tf-Ga transport into the brain enabling it to target TfR-bearing glioblastoma. We show that U-87 MG and D54 glioblastoma cell lines and multiple glioblastoma stem cell (GSC) lines express TfRs, and that their growth is inhibited by gallium maltolate (GaM) in vitro After 24 hours of incubation with GaM, cells displayed a loss of mitochondrial reserve capacity followed by a dose-dependent decrease in oxygen consumption and a decrease in the activity of the iron-dependent M2 subunit of ribonucleotide reductase (RRM2). IHC staining of rat and human tumor-bearing brains showed that glioblastoma, but not normal glial cells, expressed TfR1 and RRM2, and that glioblastoma expressed greater levels of H- and L-ferritin than normal brain. In an orthotopic U-87 MG glioblastoma xenograft rat model, GaM retarded the growth of brain tumors relative to untreated control (P = 0.0159) and reduced tumor mitotic figures (P = 0.045). Tumors in GaM-treated animals displayed an upregulation of TfR1 expression relative to control animals, thus indicating that gallium produced tumor iron deprivation. GaM also inhibited iron uptake and upregulated TfR1 expression in U-87 MG and D54 cells in vitro We conclude that GaM enters the brain via TfR1 on BMECs and targets iron metabolism in glioblastoma in vivo, thus inhibiting tumor growth. Further development of novel gallium compounds for brain tumor treatment is warranted. Mol Cancer Ther; 17(6); 1240-50. ©2018 AACR.

Synaptic connectivity between gliomas and adjacent brain was recently implicated in tumorigenesis. However, the interaction of synapse-related genes (SRGs) with patient survival and with established clinical and molecular glioma subtypes merit further study in a large patient cohort. We characterized differential expression of SRGs in gliomas and investigated SRG expression as putative clinical biomarkers in a large glioma cohort. Expression of 1189 SRGs was interrogated via RNA sequencing analysis in 603 gliomas (LGG n=451, GBM n=152) of The Cancer Genome Atlas. SRG expression patterns partitioned gliomas into two clusters that were more distinctive than priorly identified glioma subtypes. The two glioma clusters showed significantly low (14.9 months) or high median survival (105.1 months; Hazard Ratio = 7.1, 95% CI: [5.32, 9.78], p = 1.29e-46 using a log-rank test). The high survival cluster showed overrepresentation of known pro-neural and neural subtypes and IDH-mutated gliomas (p Our analysis shows that gliomas can be distinctively clustered by differential SRGs expression, suggesting that synapse-related proteins may contribute to tumorigenesis via multiple mechanisms. Furthermore, the potential utility of SRGs as clinical glioma biomarkers is supported by our creation of a prognostic SGS. Future studies elucidating interactions between tumorigenesis and synaptic mechanisms may reveal additional insights for glioma biology and therapeutic targeting.

// Ninh B. Doan 1, 2 , Hisham Alhajala 3 , Mona M. Al-Gizawiy 4 , Wade M. Mueller 2 , Scott D. Rand 4 , Jennifer M. Connelly 5 , Elizabeth J. Cochran 6 , Christopher R. Chitambar 3 , Paul Clark 7 , John Kuo 7 , Kathleen M. Schmainda 3, 8 and Shama P. Mirza 1, 9, 10 1 Biotechnology and Bioengineering Center, Medical College of Wisconsin, Milwaukee, Wisconsin, 53226, USA 2 Department of Neurosurgery, Medical College of Wisconsin, Milwaukee, Wisconsin, 53226, USA 3 Medicine, Hematology/Oncology, Medical College of Wisconsin, Milwaukee, Wisconsin, 53226, USA 4 Radiology, Medical College of Wisconsin, Milwaukee, Wisconsin, 53226, USA 5 Neurology, Medical College of Wisconsin, Milwaukee, Wisconsin, 53226, USA 6 Pathology, Medical College of Wisconsin, Milwaukee, Wisconsin, 53226, USA 7 Department of Neurological Surgery and Human Oncology, University of Wisconsin, Madison, Wisconsin, 53792, USA 8 Biophysics, Medical College of Wisconsin, Milwaukee, Wisconsin, 53226, USA 9 Obstetrics and Gynecology, Medical College of Wisconsin, Milwaukee, Wisconsin, 53226, USA 10 Department of Chemistry and Biochemistry, University of Wisconsin, Milwaukee, Wisconsin, 53211, USA Correspondence to: Shama P. Mirza, email: mirza@uwm.edu Keywords: glioblastoma, acid ceramidase, carmofur, glioblastoma cancer stem cells (GSCs), overall survival Received: December 28, 2016 Accepted: September 30, 2017 Published: November 07, 2017 ABSTRACT Glioblastoma remains the most common, malignant primary cancer of the central nervous system with a low life expectancy and an overall survival of less than 1.5 years. The treatment options are limited and there is no cure. Moreover, almost all patients develop recurrent tumors, which typically are more aggressive. Therapeutically resistant glioblastoma or glioblastoma stem-like cells (GSCs) are hypothesized to cause this inevitable recurrence. Identifying prognostic biomarkers of glioblastoma will potentially advance knowledge about glioblastoma tumorigenesis and enable discovery of more effective therapies. Proteomic analysis of more than 600 glioblastoma-specific proteins revealed, for the first time, that expression of acid ceramidase (ASAH1) is associated with poor glioblastoma survival. CD133+ GSCs express significantly higher ASAH1 compared to CD133- GSCs and serum-cultured glioblastoma cell lines, such as U87MG. These findings implicate ASAH1 as a plausible independent prognostic marker, providing a target for a therapy tailored toward GSCs. We further demonstrate that ASAH1 inhibition increases cellular ceramide level and induces apoptosis. Strikingly, U87MG cells, and three different patient-derived glioblastoma stem-like cancer cell lines were efficiently killed, through apoptosis, by three different known ASAH1 inhibitors with IC50’s ranging from 11–104 μM. In comparison, the standard glioblastoma chemotherapy agent, temozolomide, had minimal GSC-targeted effects at comparable or even higher concentrations (IC50 > 750 μM against GSCs). ASAH1 is identified as a de novo glioblastoma drug target, and ASAH1 inhibitors, such as carmofur, are shown to be highly effective and to specifically target glioblastoma GSCs. Carmofur is an ASAH1 inhibitor that crosses the blood-brain barrier, a major bottleneck in glioblastoma treatment. It has been approved in Japan since 1981 for colorectal cancer therapy. Therefore, it is poised for repurposing and translation to glioblastoma clinical trials.

INTRODUCTION: Variations in tumor volume segmentation methods in targeted radionuclide therapy (TRT) may lead to dosimetric uncertainties. This work investigates the impact of PET and MRI threshold-based tumor segmentation on TRT dosimetry in patients with primary and metastatic brain tumors. METHODS: In this study, PET/CT images of five brain cancer patients were acquired at 6, 24, and 48 hrs post-injection of (124)I-CLR1404. The tumor volume was segmented using two standardized uptake value (SUV) threshold levels, two tumor-to-background ratio (TBR) threshold levels, and a T1 Gadolinium-enhanced MRI threshold. The Dice similarity coefficient (DSC), Jaccard similarity coefficient (JSC), and overlap volume (OV) metrics were calculated to compare differences in the MRI and PET contours. The therapeutic (131)I-CLR1404 voxel-level dose distribution was calculated from the (124)I-CLR1404 activity distribution using RAPID, a Geant4 Monte Carlo internal dosimetry platform. RESULTS: The TBR, SUV, and MRI tumor volumes ranged from 2.3–63.9 cc, 0.1–34.7 cc, and 0.4–11.8 cc, respectively. The average ± standard deviation (range) was 0.19 ± 0.13 (0.01–0.51), 0.30 ± 0.17 (0.03–0.67), and 0.75 ± 0.29 (0.05–1.00) for the JSC, DSC, and OV, respectively. The DSC and JSC values were small and the OV values were large for both the MRI-SUV and MRI-TBR combinations because the regions of PET uptake were generally larger than the MRI enhancement. Notable differences in the tumor dose volume histograms (DVHs) were observed for each patient. The mean (standard deviation) (131)I-CLR1404 tumor doses ranged from 0.28–1.75 Gy/GBq (0.07–0.37 Gy/GBq). The ratio of maximum-to-minimum mean doses for each patient ranged from 1.4–2.0. CONCLUSIONS: The tumor volume and the interpretation of the tumor dose is highly sensitive to the imaging modality, PET enhancement metric, and threshold level used for tumor volume segmentation. The large variations in tumor doses clearly demonstrate the need for standard protocols for multimodality tumor segmentation in TRT dosimetry.

Objective:The aim of this study was to examine the challenges confronting surgeons performing basic science research in today's academic surgery environment.Summary of Background Data:Multiple studies have identified challenges confronting surgeon-scientists and impacting their ability to be success

OBJECTIVEGlioblastoma multiforme (GBM) is an aggressive brain cancer with median survival of less than 2 years with current treatment. Glioblastomas exhibit extensive intratumoral and interpatient heterogeneity, suggesting that successful therapies should produce broad anticancer activities. Therefore, the natural nontoxic pleiotropic agent, resveratrol, was studied for antitumorigenic effects against GBM.METHODSResveratrol's effects on cell proliferation, sphere-forming ability, and invasion were tested using multiple patient-derived GBM stem-like cell (GSC) lines and established U87 glioma cells, and changes in oncogenic AKT and tumor suppressive p53 were analyzed. Resveratrol was also tested in vivo against U87 glioma flank xenografts in mice by using multiple delivery methods, including direct tumor injection. Finally, resveratrol was delivered directly to brain tissue to determine toxicity and achievable drug concentrations in the brain parenchyma.RESULTSResveratrol significantly inhibited proliferation in U87 glioma and multiple patient-derived GSC lines, demonstrating similar inhibitory concentrations across these phenotypically heterogeneous lines. Resveratrol also inhibited the sphere-forming ability suggesting anti–stem cell effects. Additionally, resveratrol blocked U87 glioma and GSC invasion in an in vitro Matrigel Transwell assay at doses similar to those mediating antiproliferative effects. In U87 glioma cells and GSCs, resveratrol reduced AKT phosphorylation and induced p53 expression and activation that led to transcription of downstream p53 target genes. Resveratrol administration via oral gavage or ad libitum in the water supply significantly suppressed GBM xenograft growth; intratumoral or peritumoral resveratrol injection further suppressed growth and approximated tumor regression. Intracranial resveratrol injection resulted in 100-fold higher local drug concentration compared with intravenous delivery, and with no apparent toxicity.CONCLUSIONSResveratrol potently inhibited GBM and GSC growth and infiltration, acting partially via AKT deactivation and p53 induction, and suppressed glioblastoma growth in vivo. The ability of resveratrol to modulate AKT and p53, as well as reportedly many other antitumorigenic pathways, is attractive for therapy against a genetically heterogeneous tumor such as GBM. Although resveratrol exhibits low bioavailability when administered orally or intravenously, novel delivery methods such as direct injection (i.e., convection-enhanced delivery) could potentially be used to achieve and maintain therapeutic doses in the brain. Resveratrol's nontoxic nature and broad anti-GBM effects make it a compelling candidate to supplement current GBM therapies.

A recent Phase 3 study of newly diagnosed glioblastoma (GBM) demonstrated the addition of Tumor Treating Fields (TTFields) to temozolomide (TMZ) after combined radiation/TMZ significantly increased survival and progression free survival. Preliminary data suggested benefit with both methylated and unmethylated O-6-methylguanine-DNA methyl-transferase (MGMT) promoter status. To date, however, there have been no studies to address the potential interactions of TTFields and TMZ. Thus, the effects of TTFields and TMZ were studied in vitro using patient-derived GBM stem-like cells (GSCs) including MGMT expressing (TMZ resistant:12.1 and 22 GSC) and non-MGMT expressing (TMZ sensitive:33 and 114 GSC) lines. Dose-response curves were constructed using cell proliferation and sphere-forming assays. Results demonstrated a ≥10-fold increase in TMZ resistance of MGMT-expressing (12.1 GSCs: IC50=160 μM; 22 GSCs: IC50=44 μM) compared to MGMT non-expressing (33 GSCs: IC50=1.5 μM; 114 GSCs: IC50=5.2 μM) lines. TTFields inhibited 12.1 GSC proliferation at all tested doses (50-500 kHz) with an optimal frequency of 200 kHz. At 200 kHz, TTFields inhibited proliferation and tumor sphere formation of both MGMT GSC subtypes at comparable levels (12.1 GSC: 74±2.9% and 38±3.2%, respectively; 22 GSC: 61±11% and 38±2.6%, respectively; 33 GSC: 56±9.5% and 60±7.1%, respectively; 114 GSC: 79± 3.5% and 41±4.3%, respectively). In combination, TTFields (200 kHz) and TMZ showed an additive anti-neoplastic effect with equal efficacy for TTFields in both cell types (i.e., +/- MGMT expression) with no effect on TMZ resistance. This is the first demonstration of the effects of TTFields on cancer stem cells. The expansion of such studies may have clinical implications.

Over the past two decades, synergistic innovations in imaging technology have resulted in a revolution in which a range of biomedical applications are now benefiting from fluorescence imaging. Specifically, advances in fluorophore chemistry and imaging hardware, and the identification of targetable biomarkers have now positioned intraoperative fluorescence as a highly specific real-time detection modality for surgeons in oncology. In particular, the deeper tissue penetration and limited autofluorescence of near-infrared (NIR) fluorescence imaging improves the translational potential of this modality over visible-light fluorescence imaging. Rapid developments in fluorophores with improved characteristics, detection instrumentation, and targeting strategies led to the clinical testing in the early 2010s of the first targeted NIR fluorophores for intraoperative cancer detection. The foundations for the advances that underline this technology continue to be nurtured by the multidisciplinary collaboration of chemists, biologists, engineers, and clinicians. In this Review, we highlight the latest developments in NIR fluorophores, cancer-targeting strategies, and detection instrumentation for intraoperative cancer detection, and consider the unique challenges associated with their effective application in clinical settings.

INTRODUCTION The median survival of glioblastoma (GBM) patients remains less than two years even with state-of-the-art treatment. Current targeted GBM therapies demonstrate initial therapeutic benefit; however, patients relapse due to therapeutic selection of treatment resistant GBM cellular populations. Therefore, we propose targeting pathologic disruption of the blood brain barrier (BBB) via exposure of neural ECM, rather than disease markers, to overcome therapy-resistant GBM. METHODS We identify Variable Lymphocyte Receptors (VLRs, the antigen recognition system used by lamprey) that demonstrate high specificity for neural ECM. Candidate VLRs underwent further refinement using in vitro binding assays and ex vivo tissue staining. Utilizing pathologic disruption of BBB as an approach for targeting GBM was confirmed in vivo using intracranial murine glioblastoma models. RESULTS The lead neural ECM-binding VLR candidate, named P1C10, demonstrates diffuse binding to parenchymal neural ECM, without detectable binding to other tissues. P1C10 demonstrates nanomolar affinity for in vitro derived neural ECM, and preferentially accumulates at intracranial GL261 and U87 lesions in murine GBM models. Finally, administration of P1C10-targeted doxorubicin-loaded liposomes significantly extends the survival of mice bearing intracranial U87 GBM. CONCLUSIONS We identified VLRs that bind neural ECM, and demonstrate their utility for delivering compounds and nanoparticles to sites of GBM induced blood brain barrier disruption. This novel strategy allows for targeting therapeutics via the underlying physiology of GBM rather than relying on cellular disease markers that are often lost in patients that relapse after targeting therapies.

Cerebral aneurysm clips are biomedical implants applied by neurosurgeons to re-approximate arterial vessel walls and prevent catastrophic aneurysmal hemorrhages in patients. Current methods of aneurysm clip production are labor intensive and time-consuming, leading to high costs per implant and limited variability in clip morphology. Metal additive manufacturing is investigated as an alternative to traditional manufacturing methods that may enable production of patient-specific aneurysm clips to account for variations in individual vascular anatomy and possibly reduce surgical complication risks. Relevant challenges to metal additive manufacturing are investigated for biomedical implants, including material choice, design limitations, postprocessing, printed material properties, and combined production methods. Initial experiments with additive manufacturing of 316 L stainless steel aneurysm clips are carried out on a selective laser melting (SLM) system. The dimensions of the printed clips were found to be within 0.5% of the dimensions of the designed clips. Hardness and density of the printed clips (213 ± 7 HV1 and 7.9 g/cc, respectively) were very close to reported values for 316 L stainless steel, as expected. No ferrite and minimal porosity is observed in a cross section of a printed clip, with some anisotropy in the grain orientation. A clamping force of approximately 1 N is measured with a clip separation of 1.5 mm. Metal additive manufacturing shows promise for use in the creation of custom aneurysm clips, but some of the challenges discussed will need to be addressed before clinical use is possible.

This study demonstrates targeting of blood-brain barrier disruption to deliver payloads at sites of neurological disease., Diseases that lead to blood-brain barrier (BBB) disruption will pathologically expose normally inaccessible brain extracellular matrix (ECM) to circulating blood components. Therefore, we hypothesized that brain ECM-targeting moieties could specifically target the disrupted BBB and potentially deliver therapies. Variable lymphocyte receptors (VLRs) that preferentially associate with brain ECM were identified from an immune VLR library via yeast surface display biopanning coupled with a moderate throughput ECM screen. Brain ECM binding of VLR clones to murine and human brain tissue sections was confirmed. After systemic administration, P1C10, the lead brain ECM-targeting VLR candidate, specifically accumulated in brains with mannitol-disrupted BBB and at disrupted BBB regions in two different intracranial glioblastoma models. We also demonstrate P1C10’s ability to deliver doxorubicin-loaded liposomes, leading to significantly improved survival in glioblastoma-bearing mice. Thus, VLRs can be used to selectively target pathologically exposed brain ECM and deliver drug payloads.