Novel immunotherapeutics against LGR5 to target multiple cancer types.

We have developed and validated a highly specific, versatile antibody to the extracellular domain of human LGR5 (α-LGR5). α-LGR5 detects LGR5 overexpression in >90% of colorectal cancer (CRC), hepatocellular carcinoma (HCC) and pre-B-ALL tumour cells and was used to generate an Antibody-Drug Conjugate (α-LGR5-ADC), Bispecific T-cell Engager (α-LGR5-BiTE) and Chimeric Antigen Receptor (α-LGR5-CAR). α-LGR5-ADC was the most effective modality for targeting LGR5+ cancer cells in vitro and demonstrated potent anti-tumour efficacy in a murine model of human NALM6 pre-B-ALL driving tumour attrition to less than 1% of control treatment. α-LGR5-BiTE treatment was less effective in the pre-B-ALL cancer model yet promoted a twofold reduction in tumour burden. α-LGR5-CAR-T cells also showed specific and potent LGR5+ cancer cell killing in vitro and effective tumour targeting with a fourfold decrease in pre-B-ALL tumour burden relative to controls. Taken together, we show that α-LGR5 can not only be used as a research tool and a biomarker but also provides a versatile building block for a highly effective immune therapeutic portfolio targeting a range of LGR5-expressing cancer cells.

Deuterium Metabolic Imaging Differentiates Glioblastoma Metabolic Subtypes and Detects Early Response to Chemoradiotherapy.

Metabolic subtypes of glioblastoma (GBM) have different prognoses and responses to treatment. Deuterium metabolic imaging with 2H-labeled substrates is a potential approach to stratify patients into metabolic subtypes for targeted treatment. In this study, we used 2H magnetic resonance spectroscopy and magnetic resonance spectroscopic imaging (MRSI) measurements of [6,6'-2H2]glucose metabolism to identify metabolic subtypes and their responses to chemoradiotherapy in patient-derived GBM xenografts in vivo. The metabolism of patient-derived cells was first characterized in vitro by measuring the oxygen consumption rate, a marker of mitochondrial tricarboxylic acid cycle activity, as well as the extracellular acidification rate and 2H-labeled lactate production from [6,6'-2H2]glucose, which are markers of glycolytic activity. Two cell lines representative of a glycolytic subtype and two representative of a mitochondrial subtype were identified. 2H magnetic resonance spectroscopy and MRSI measurements showed similar concentrations of 2H-labeled glucose from [6,6'-2H2]glucose in all four tumor models when implanted orthotopically in mice. The glycolytic subtypes showed higher concentrations of 2H-labeled lactate than the mitochondrial subtypes and normal-appearing brain tissue, whereas the mitochondrial subtypes showed more glutamate/glutamine labeling, a surrogate for tricarboxylic acid cycle activity, than the glycolytic subtypes and normal-appearing brain tissue. The response of the tumors to chemoradiation could be detected within 24 hours of treatment completion, with the mitochondrial subtypes showing a decrease in both 2H-labeled glutamate/glutamine and lactate concentrations and glycolytic tumors showing a decrease in 2H-labeled lactate concentration. This technique has the potential to be used clinically for treatment selection and early detection of treatment response. SIGNIFICANCE: Deuterium magnetic resonance spectroscopic imaging of glucose metabolism has the potential to differentiate between glycolytic and mitochondrial metabolic subtypes in glioblastoma and to evaluate early treatment responses, which could guide patient treatment.

Molecular diversity in isocitrate dehydrogenase-wild-type glioblastoma.

In the dynamic landscape of glioblastoma, the 2021 World Health Organization Classification of Central Nervous System tumours endeavoured to establish biological homogeneity, yet isocitrate dehydrogenase-wild-type (IDH-wt) glioblastoma persists as a tapestry of clinical and molecular diversity. Intertumoural heterogeneity in IDH-wt glioblastoma presents a formidable challenge in treatment strategies. Recent strides in genetics and molecular biology have enhanced diagnostic precision, revealing distinct subtypes and invasive patterns that influence survival in patients with IDH-wt glioblastoma. Genetic and molecular biomarkers, such as the overexpression of neurofibromin 1, phosphatase and tensin homolog and/or cyclin-dependent kinase inhibitor 2A, along with specific immune cell abundance and neurotransmitters, correlate with favourable outcomes. Conversely, increased expression of epidermal growth factor receptor tyrosine kinase, platelet-derived growth factor receptor alpha and/or vascular endothelial growth factor receptor, coupled with the prevalence of glioma stem cells, tumour-associated myeloid cells, regulatory T cells and exhausted effector cells, signifies an unfavourable prognosis. The methylation status of O6-methylguanine-DNA methyltransferase and the influence of microenvironmental factors and neurotransmitters further shape treatment responses. Understanding intertumoural heterogeneity is complemented by insights into intratumoural dynamics and cellular interactions within the tumour microenvironment. Glioma stem cells and immune cell composition significantly impact progression and outcomes, emphasizing the need for personalized therapies targeting pro-tumoural signalling pathways and resistance mechanisms. A successful glioblastoma management demands biomarker identification, combination therapies and a nuanced approach considering intratumoural variability. These advancements herald a transformative era in glioblastoma comprehension and treatment.

The role of branched-chain aminotransferase 1 in driving glioblastoma cell proliferation and invasion varies with tumor subtype.

Background: Branched-chain aminotransferase 1 (BCAT1) has been proposed to drive proliferation and invasion of isocitrate dehydrogenase (IDH) wild-type glioblastoma cells. However, the Cancer Genome Atlas (TCGA) dataset shows considerable variation in the expression of this enzyme in glioblastoma. The aim of this study was to determine the role of BCAT1 in driving the proliferation and invasion of glioblastoma cells and xenografts that have widely differing levels of BCAT1 expression and the mechanism responsible. Methods: The activity of BCAT1 was modulated in IDH wild-type patient-derived glioblastoma cell lines, and in orthotopically implanted tumors derived from these cells, to examine the effects of BCAT1 expression on tumor phenotype. Results: In cells with constitutively high BCAT1 expression and a glycolytic metabolic phenotype, inducible shRNA knockdown of the enzyme resulted in reduced proliferation and invasion by increasing the concentration of α-ketoglutarate, leading to reduced DNA methylation, HIF-1α destabilization, and reduced expression of the transcription factor Forkhead box protein M1 (FOXM1). Conversely, overexpression of the enzyme increased HIF-1α expression and promoted proliferation and invasion. However, in cells with an oxidative phenotype and very low constitutive expression of BCAT1 increased expression of the enzyme had no effect on invasion and reduced cell proliferation. This occurred despite an increase in HIF-1α levels and could be explained by decreased TCA cycle flux. Conclusions: There is a wide variation in BCAT1 expression in glioblastoma and its role in proliferation and invasion is dependent on tumor subtype.

Personalised therapeutic approaches to glioblastoma: A systematic review.

Introduction: Glioblastoma is the most common and malignant primary brain tumour with median survival of 14.6 months. Personalised medicine aims to improve survival by targeting individualised patient characteristics. However, a major limitation has been application of targeted therapies in a non-personalised manner without biomarker enrichment. This has risked therapies being discounted without fair and rigorous evaluation. The objective was therefore to synthesise the current evidence on survival efficacy of personalised therapies in glioblastoma. Methods: Studies reporting a survival outcome in human adults with supratentorial glioblastoma were eligible. PRISMA guidelines were followed. MEDLINE, Embase, Scopus, Web of Science and the Cochrane Library were searched to 5th May 2022. Clinicaltrials.gov was searched to 25th May 2022. Reference lists were hand-searched. Duplicate title/abstract screening, data extraction and risk of bias assessments were conducted. A quantitative synthesis is presented. Results: A total of 102 trials were included: 16 were randomised and 41 studied newly diagnosed patients. Of 5,527 included patients, 59.4% were male and mean age was 53.7 years. More than 20 types of personalised therapy were included: targeted molecular therapies were the most studied (33.3%, 34/102), followed by autologous dendritic cell vaccines (32.4%, 33/102) and autologous tumour vaccines (10.8%, 11/102). There was no consistent evidence for survival efficacy of any personalised therapy. Conclusion: Personalised glioblastoma therapies remain of unproven survival benefit. Evidence is inconsistent with high risk of bias. Nonetheless, encouraging results in some trials provide reason for optimism. Future focus should address target-enriched trials, combination therapies, longitudinal biomarker monitoring and standardised reporting.

A Prospective Study of Longitudinal Risks of Cognitive Deficit for People Undergoing Glioblastoma Surgery Using a Tablet Computer Cognition Testing Battery: Towards Personalized Understanding of Risks to Cognitive Function.

Glioblastoma and the surgery to remove it pose high risks to the cognitive function of patients. Little reliable data exist about these risks, especially postoperatively before radiotherapy. We hypothesized that cognitive deficit risks detected before surgery will be exacerbated by surgery in patients with glioblastoma undergoing maximal treatment regimens. We used longitudinal electronic cognitive testing perioperatively to perform a prospective, longitudinal, observational study of 49 participants with glioblastoma undergoing surgery. Before surgery (A1), the participant risk of deficit in 5/6 cognitive domains was increased compared to normative data. Of these, the risks to Attention (OR = 31.19), Memory (OR = 97.38), and Perception (OR = 213.75) were markedly increased. These risks significantly increased in the early period after surgery (A2) when patients were discharged home or seen in the clinic to discuss histology results. For participants tested at 4-6 weeks after surgery (A3) before starting radiotherapy, there was evidence of risk reduction towards A1. The observed risks of cognitive deficit were independent of patient-specific, tumour-specific, and surgery-specific co-variates. These results reveal a timeframe of natural recovery in the first 4-6 weeks after surgery based on personalized deficit profiles for each participant. Future research in this period could investigate personalized rehabilitation tools to aid the recovery process found.

Targeting succinate metabolism to decrease brain injury upon mechanical thrombectomy treatment of ischemic stroke.

Current treatments for acute ischemic stroke aim to reinstate a normal perfusion in the ischemic territory but can also cause significant ischemia-reperfusion (IR) injury. Previous data in experimental models of stroke show that ischemia leads to the accumulation of succinate, and, upon reperfusion, the accumulated succinate is rapidly oxidized by succinate dehydrogenase (SDH) to drive superoxide production at mitochondrial complex I. Despite this process initiating IR injury and causing further tissue damage, the potential of targeting succinate metabolism to minimize IR injury remains unexplored. Using both quantitative and untargeted high-resolution metabolomics, we show a time-dependent accumulation of succinate in both human and mouse brain exposed to ischemia ex vivo. In a mouse model of ischemic stroke/mechanical thrombectomy mass spectrometry imaging (MSI) shows that succinate accumulation is confined to the ischemic region, and that the accumulated succinate is rapidly oxidized upon reperfusion. Targeting succinate oxidation by systemic infusion of the SDH inhibitor malonate upon reperfusion leads to a dose-dependent decrease in acute brain injury. Together these findings support targeting succinate metabolism upon reperfusion to decrease IR injury as a valuable adjunct to mechanical thrombectomy in ischemic stroke.

Cell-Free Mitochondrial DNA in Acute Brain Injury.

Traumatic brain injury and aneurysmal subarachnoid haemorrhage are a major cause of morbidity and mortality worldwide. Treatment options remain limited and are hampered by our understanding of the cellular and molecular mechanisms, including the inflammatory response observed in the brain. Mitochondrial DNA (mtDNA) has been shown to activate an innate inflammatory response by acting as a damage-associated molecular pattern (DAMP). Here, we show raised circulating cell-free (ccf) mtDNA levels in both cerebrospinal fluid (CSF) and serum within 48 h of brain injury. CSF ccf-mtDNA levels correlated with clinical severity and the interleukin-6 cytokine response. These findings support the use of ccf-mtDNA as a biomarker after acute brain injury linked to the inflammatory disease mechanism.

Myeloid cell interferon secretion restricts Zika flavivirus infection of developing and malignant human neural progenitor cells.

Zika virus (ZIKV) can infect human developing brain (HDB) progenitors resulting in epidemic microcephaly, whereas analogous cellular tropism offers treatment potential for the adult brain cancer, glioblastoma (GBM). We compared productive ZIKV infection in HDB and GBM primary tissue explants that both contain SOX2+ neural progenitors. Strikingly, although the HDB proved uniformly vulnerable to ZIKV infection, GBM was more refractory, and this correlated with an innate immune expression signature. Indeed, GBM-derived CD11b+ microglia/macrophages were necessary and sufficient to protect progenitors against ZIKV infection in a non-cell autonomous manner. Using SOX2+ GBM cell lines, we found that CD11b+-conditioned medium containing type 1 interferon beta (IFNß) promoted progenitor resistance to ZIKV, whereas inhibition of JAK1/2 signaling restored productive infection. Additionally, CD11b+ conditioned medium, and IFNß treatment rendered HDB progenitor lines and explants refractory to ZIKV. These findings provide insight into neuroprotection for HDB progenitors as well as enhanced GBM oncolytic therapies.

Imaging Glioblastoma Metabolism by Using Hyperpolarized [1-13C]Pyruvate Demonstrates Heterogeneity in Lactate Labeling: A Proof of Principle Study.

Purpose To evaluate glioblastoma (GBM) metabolism by using hyperpolarized carbon 13 (13C) MRI to monitor the exchange of the hyperpolarized 13C label between injected [1-13C]pyruvate and tumor lactate and bicarbonate. Materials and Methods In this prospective study, seven treatment-naive patients (age [mean ± SD], 60 years ± 11; five men) with GBM were imaged at 3 T by using a dual-tuned 13C-hydrogen 1 head coil. Hyperpolarized [1-13C]pyruvate was injected, and signal was acquired by using a dynamic MRI spiral sequence. Metabolism was assessed within the tumor, in the normal-appearing brain parenchyma (NABP), and in healthy volunteers by using paired or unpaired t tests and a Wilcoxon signed rank test. The Spearman ρ correlation coefficient was used to correlate metabolite labeling with lactate dehydrogenase A (LDH-A) expression and some immunohistochemical markers. The Benjamini-Hochberg procedure was used to correct for multiple comparisons. Results The bicarbonate-to-pyruvate (BP) ratio was lower in the tumor than in the contralateral NABP (P < .01). The tumor lactate-to-pyruvate (LP) ratio was not different from that in the NABP (P = .38). The LP and BP ratios in the NABP were higher than those observed previously in healthy volunteers (P < .05). Tumor lactate and bicarbonate signal intensities were strongly correlated with the pyruvate signal intensity (ρ = 0.92, P < .001, and ρ = 0.66, P < .001, respectively), and the LP ratio was weakly correlated with LDH-A expression in biopsy samples (ρ = 0.43, P = .04). Conclusion Hyperpolarized 13C MRI demonstrated variation in lactate labeling in GBM, both within and between tumors. In contrast, bicarbonate labeling was consistently lower in tumors than in the surrounding NABP. Keywords: Hyperpolarized 13C MRI, Glioblastoma, Metabolism, Cancer, MRI, Neuro-oncology Supplemental material is available for this article. Published under a CC BY 4.0 license.

Cell-free DNA technologies for the analysis of brain cancer.

Survival for glioma patients has shown minimal improvement over the past 20 years. The ability to detect and monitor gliomas relies primarily upon imaging technologies that lack sensitivity and specificity, especially during the post-surgical treatment phase. Treatment-response monitoring with an effective liquid-biopsy paradigm may also provide the most facile clinical scenario for liquid-biopsy integration into brain-tumour care. Conceptually, liquid biopsy is advantageous when compared with both tissue sampling (less invasive) and imaging (more sensitive and specific), but is hampered by technical and biological problems. These problems predominantly relate to low concentrations of tumour-derived DNA in the bloodstream of glioma patients. In this review, we highlight methods by which the neuro-oncological scientific and clinical communities have attempted to circumvent this limitation. The use of novel biological, technological and computational approaches will be explored. The utility of alternate bio-fluids, tumour-guided sequencing, epigenomic and fragmentomic methods may eventually be leveraged to provide the biological and technological means to unlock a wide range of clinical applications for liquid biopsy in glioma.

Mechanically matching the rheological properties of brain tissue for drug-delivery in human glioblastoma models.

Peptide functionalized hyaluronic acid (HACF) cross-linked by cucurbit[8]uril (CB[8]), a new class of drug-delivery reservoirs, is used to enable improved drug bioavailability for glioblastoma tumors in patient-derived xenograft (PDX) models. The mechanical and viscoelastic properties of native human and mouse tissues are measured over 8 h via oscillatory rheology under physiological conditions. Treatment with drug-loaded hydrogels allowed for a significant survival impact of 45 % (55.5-80.5 days). A relationship between the type of PDX tumor formed-a consequence of the heterogeneic nature of GB tumors-and changes in the initial survival is observed owing to greater local pressure from stiffer tumors. These biocompatible and tailorable materials warrant use as drug delivery reservoirs in PDX resection models, where the mechanical properties can be readily adjusted to match the stiffness of local tissue and thus have potential to improve the survival of GB patients.

Fragmentation patterns and personalized sequencing of cell-free DNA in urine and plasma of glioma patients.

Glioma-derived cell-free DNA (cfDNA) is challenging to detect using liquid biopsy because quantities in body fluids are low. We determined the glioma-derived DNA fraction in cerebrospinal fluid (CSF), plasma, and urine samples from patients using sequencing of personalized capture panels guided by analysis of matched tumor biopsies. By sequencing cfDNA across thousands of mutations, identified individually in each patient's tumor, we detected tumor-derived DNA in the majority of CSF (7/8), plasma (10/12), and urine samples (10/16), with a median tumor fraction of 6.4 × 10-3 , 3.1 × 10-5 , and 4.7 × 10-5 , respectively. We identified a shift in the size distribution of tumor-derived cfDNA fragments in these body fluids. We further analyzed cfDNA fragment sizes using whole-genome sequencing, in urine samples from 35 glioma patients, 27 individuals with non-malignant brain disorders, and 26 healthy individuals. cfDNA in urine of glioma patients was significantly more fragmented compared to urine from patients with non-malignant brain disorders (P = 1.7 × 10-2 ) and healthy individuals (P = 5.2 × 10-9 ). Machine learning models integrating fragment length could differentiate urine samples from glioma patients (AUC = 0.80-0.91) suggesting possibilities for truly non-invasive cancer detection.

A map of transcriptional heterogeneity and regulatory variation in human microglia.

Microglia, the tissue-resident macrophages of the central nervous system (CNS), play critical roles in immune defense, development and homeostasis. However, isolating microglia from humans in large numbers is challenging. Here, we profiled gene expression variation in primary human microglia isolated from 141 patients undergoing neurosurgery. Using single-cell and bulk RNA sequencing, we identify how age, sex and clinical pathology influence microglia gene expression and which genetic variants have microglia-specific functions using expression quantitative trait loci (eQTL) mapping. We follow up one of our findings using a human induced pluripotent stem cell-based macrophage model to fine-map a candidate causal variant for Alzheimer's disease at the BIN1 locus. Our study provides a population-scale transcriptional map of a critically important cell for human CNS development and disease.

ctDNA monitoring using patient-specific sequencing and integration of variant reads.

Circulating tumor-derived DNA (ctDNA) can be used to monitor cancer dynamics noninvasively. Detection of ctDNA can be challenging in patients with low-volume or residual disease, where plasma contains very few tumor-derived DNA fragments. We show that sensitivity for ctDNA detection in plasma can be improved by analyzing hundreds to thousands of mutations that are first identified by tumor genotyping. We describe the INtegration of VAriant Reads (INVAR) pipeline, which combines custom error-suppression methods and signal-enrichment approaches based on biological features of ctDNA. With this approach, the detection limit in each sample can be estimated independently based on the number of informative reads sequenced across multiple patient-specific loci. We applied INVAR to custom hybrid-capture sequencing data from 176 plasma samples from 105 patients with melanoma, lung, renal, glioma, and breast cancer across both early and advanced disease. By integrating signal across a median of >105 informative reads, ctDNA was routinely quantified to 1 mutant molecule per 100,000, and in some cases with high tumor mutation burden and/or plasma input material, to parts per million. This resulted in median area under the curve (AUC) values of 0.98 in advanced cancers and 0.80 in early-stage and challenging settings for ctDNA detection. We generalized this method to whole-exome and whole-genome sequencing, showing that INVAR may be applied without requiring personalized sequencing panels so long as a tumor mutation list is available. As tumor sequencing becomes increasingly performed, such methods for personalized cancer monitoring may enhance the sensitivity of cancer liquid biopsies.

Measurement of Plasma Cell-Free Mitochondrial Tumor DNA Improves Detection of Glioblastoma in Patient-Derived Orthotopic Xenograft Models.

The factors responsible for the low detection rate of cell-free tumor DNA (ctDNA) in the plasma of patients with glioblastoma (GBM) are currently unknown. In this study, we measured circulating nucleic acids in patient-derived orthotopically implanted xenograft (PDOX) models of GBM (n = 64) and show that tumor size and cell proliferation, but not the integrity of the blood-brain barrier or cell death, affect the release of ctDNA in treatment-naïve GBM PDOX. Analysis of fragment length profiles by shallow genome-wide sequencing (<0.2× coverage) of host (rat) and tumor (human) circulating DNA identified a peak at 145 bp in the human DNA fragments, indicating a difference in the origin or processing of the ctDNA. The concentration of ctDNA correlated with cell death only after treatment with temozolomide and radiotherapy. Digital PCR detection of plasma tumor mitochondrial DNA (tmtDNA), an alternative to detection of nuclear ctDNA, improved plasma DNA detection rate (82% vs. 24%) and allowed detection in cerebrospinal fluid and urine. Mitochondrial mutations are prevalent across all cancers and can be detected with high sensitivity, at low cost, and without prior knowledge of tumor mutations via capture-panel sequencing. Coupled with the observation that mitochondrial copy number increases in glioma, these data suggest analyzing tmtDNA as a more sensitive method to detect and monitor tumor burden in cancer, specifically in GBM, where current methods have largely failed. SIGNIFICANCE: These findings show that detection of tumor mitochondrial DNA is more sensitive than circulating tumor DNA analysis to detect and monitor tumor burden in patient-derived orthotopic xenografts of glioblastoma.

Enhanced detection of circulating tumor DNA by fragment size analysis.

Existing methods to improve detection of circulating tumor DNA (ctDNA) have focused on genomic alterations but have rarely considered the biological properties of plasma cell-free DNA (cfDNA). We hypothesized that differences in fragment lengths of circulating DNA could be exploited to enhance sensitivity for detecting the presence of ctDNA and for noninvasive genomic analysis of cancer. We surveyed ctDNA fragment sizes in 344 plasma samples from 200 patients with cancer using low-pass whole-genome sequencing (0.4×). To establish the size distribution of mutant ctDNA, tumor-guided personalized deep sequencing was performed in 19 patients. We detected enrichment of ctDNA in fragment sizes between 90 and 150 bp and developed methods for in vitro and in silico size selection of these fragments. Selecting fragments between 90 and 150 bp improved detection of tumor DNA, with more than twofold median enrichment in >95% of cases and more than fourfold enrichment in >10% of cases. Analysis of size-selected cfDNA identified clinically actionable mutations and copy number alterations that were otherwise not detected. Identification of plasma samples from patients with advanced cancer was improved by predictive models integrating fragment length and copy number analysis of cfDNA, with area under the curve (AUC) >0.99 compared to AUC <0.80 without fragmentation features. Increased identification of cfDNA from patients with glioma, renal, and pancreatic cancer was achieved with AUC > 0.91 compared to AUC < 0.5 without fragmentation features. Fragment size analysis and selective sequencing of specific fragment sizes can boost ctDNA detection and could complement or provide an alternative to deeper sequencing of cfDNA.

Detection of cell-free DNA fragmentation and copy number alterations in cerebrospinal fluid from glioma patients.

Glioma is difficult to detect or characterize using current liquid biopsy approaches. Detection of cell-free tumor DNA (cftDNA) in cerebrospinal fluid (CSF) has been proposed as an alternative to detection in plasma. We used shallow whole-genome sequencing (sWGS, at a coverage of < 0.4×) of cell-free DNA from the CSF of 13 patients with primary glioma to determine somatic copy number alterations and DNA fragmentation patterns. This allowed us to determine the presence of cftDNA in CSF without any prior knowledge of point mutations present in the tumor. We also showed that the fragmentation pattern of cell-free DNA in CSF is different from that in plasma. This low-cost screening method provides information on the tumor genome and can be used to target those patients with high levels of cftDNA for further larger-scale sequencing, such as by whole-exome and whole-genome sequencing.

[18F]fluoroethyltyrosine-induced Cerenkov Luminescence Improves Image-Guided Surgical Resection of Glioma.

The extent of surgical resection is significantly correlated with outcome in glioma; however, current intraoperative navigational tools are useful only in a subset of patients. We show here that a new optical intraoperative technique, Cerenkov luminescence imaging (CLI) following intravenous injection of O­(2-[18F]fluoroethyl)-L-tyrosine (FET), can be used to accurately delineate glioma margins, performing better than the current standard of fluorescence imaging with 5-aminolevulinic acid (5-ALA). Methods: Rats implanted orthotopically with U87, F98 and C6 glioblastoma cells were injected with FET and 5-aminolevulinic acid (5-ALA). Positive and negative tumor regions on histopathology were compared with CL and fluorescence images. The capability of FET CLI and 5-ALA fluorescence imaging to detect tumor was assessed using receptor operator characteristic curves and optimal thresholds (CLIOptROC and 5-ALAOptROC) separating tumor from healthy brain tissue were determined. These thresholds were used to guide prospective tumor resections, where the presence of tumor cells in the resected material and in the remaining brain were assessed by Ki-67 staining. Results: FET CLI signal was correlated with signal in preoperative PET images (y = 1.06x - 0.01; p < 0.0001) and with expression of the amino acid transporter SLC7A5 (LAT1). FET CLI (AUC = 97%) discriminated between glioblastoma and normal brain in human and rat orthografts more accurately than 5-ALA fluorescence (AUC = 91%), with a sensitivity >92% and specificity >91%, and resulted in a more complete tumor resection. Conclusion: FET CLI can be used to accurately delineate glioblastoma tumor margins, performing better than the current standard of fluorescence imaging following 5-ALA administration, and is therefore a promising technique for clinical translation.