The heterogeneous sensitivity of pediatric brain tumors to different oncolytic viruses is predicted by unique gene expression profiles.

Despite decades of research, the prognosis of high-grade pediatric brain tumors (PBTs) remains dismal; however, recent cases of favorable clinical responses were documented in clinical trials using oncolytic viruses (OVs). In the current study, we employed four different species of OVs: adenovirus Delta24-RGD, herpes simplex virus rQNestin34.5v1, reovirus R124, and the non-virulent Newcastle disease virus rNDV-F0-GFP against three entities of PBTs (high-grade gliomas, atypical teratoid/rhabdoid tumors, and ependymomas) to determine their in vitro efficacy. These four OVs were screened on 14 patient-derived PBT cell cultures and the degree of oncolysis was assessed using an ATP-based assay. Subsequently, the observed viral efficacies were correlated to whole transcriptome data and Gene Ontology analysis was performed. Although no significant tumor type-specific OV efficacy was observed, the analysis revealed the intrinsic biological processes that associated with OV efficacy. The predictive power of the identified expression profiles was further validated in vitro by screening additional PBTs. In summary, our results demonstrate OV susceptibility of multiple patient-derived PBT entities and the ability to predict in vitro responses to OVs using unique expression profiles. Such profiles may hold promise for future OV preselection with effective oncolytic potency in a specific tumor, therewith potentially improving OV responses.

Genomic Exploration of Distinct Molecular Phenotypes Steering Temozolomide Resistance Development in Patient-Derived Glioblastoma Cells.

Chemotherapy using temozolomide is the standard treatment for patients with glioblastoma. Despite treatment, prognosis is still poor largely due to the emergence of temozolomide resistance. This resistance is closely linked to the widely recognized inter- and intra-tumoral heterogeneity in glioblastoma, although the underlying mechanisms are not yet fully understood. To induce temozolomide resistance, we subjected 21 patient-derived glioblastoma cell cultures to Temozolomide treatment for a period of up to 90 days. Prior to treatment, the cells' molecular characteristics were analyzed using bulk RNA sequencing. Additionally, we performed single-cell RNA sequencing on four of the cell cultures to track the evolution of temozolomide resistance. The induced temozolomide resistance was associated with two distinct phenotypic behaviors, classified as "adaptive" (ADA) or "non-adaptive" (N-ADA) to temozolomide. The ADA phenotype displayed neurodevelopmental and metabolic gene signatures, whereas the N-ADA phenotype expressed genes related to cell cycle regulation, DNA repair, and protein synthesis. Single-cell RNA sequencing revealed that in ADA cell cultures, one or more subpopulations emerged as dominant in the resistant samples, whereas N-ADA cell cultures remained relatively stable. The adaptability and heterogeneity of glioblastoma cells play pivotal roles in temozolomide treatment and contribute to the tumor's ability to survive. Depending on the tumor's adaptability potential, subpopulations with acquired resistance mechanisms may arise.

Local delivery of hrBMP4 as an anticancer therapy in patients with recurrent glioblastoma: a first-in-human phase 1 dose escalation trial.

BACKGROUND: This Phase 1 study evaluates the intra- and peritumoral administration by convection enhanced delivery (CED) of human recombinant Bone Morphogenetic Protein 4 (hrBMP4) - an inhibitory regulator of cancer stem cells (CSCs) - in recurrent glioblastoma. METHODS: In a 3 + 3 dose escalation design, over four to six days, fifteen recurrent glioblastoma patients received, by CED, one of five doses of hrBMP4 ranging from 0·5 to 18 mg. Patients were followed by periodic physical, neurological, blood testing, magnetic resonance imaging (MRI) and quality of life evaluations. The primary objective of this first-in-human study was to determine the safety, dose-limiting toxicities (DLT) and maximum tolerated dose (MTD) of hrBMP4. Secondary objectives were to assess potential efficacy and systemic exposure to hrBMP4 upon intracerebral infusion. RESULTS: Intra- and peritumoral infusion of hrBMP4 was safe and well-tolerated. We observed no serious adverse events related to this drug. Neither MTD nor DLT were reached. Three patients had increased hrBMP4 serum levels at the end of infusion, which normalized within 4 weeks, without sign of toxicity. One patient showed partial response and two patients a complete (local) tumor response, which was maintained until the most recent follow-up, 57 and 30 months post-hrBMP4. Tumor growth was inhibited in areas permeated by hrBMP4. CONCLUSION: Local delivery of hrBMP4 in and around recurring glioblastoma is safe and well-tolerated. Three patients responded to the treatment. A complete response and long-term survival occurred in two of them. This warrants further clinical studies on this novel treatment targeting glioblastoma CSCs. TRIAL REGISTRATION: ClinicaTrials.gov identifier: NCT02869243.

Ex vivo drug sensitivity screening predicts response to temozolomide in glioblastoma patients and identifies candidate biomarkers.

BACKGROUND: Patient-derived glioma stem-like cells (GSCs) have become the gold-standard in neuro-oncological research; however, it remains to be established whether loss of in situ microenvironment affects the clinically-predictive value of this model. We implemented a GSC monolayer system to investigate in situ-in vitro molecular correspondence and the relationship between in vitro and patient response to temozolomide (TMZ). METHODS: DNA/RNA-sequencing was performed on 56 glioblastoma tissues and 19 derived GSC cultures. Sensitivity to TMZ was screened across 66 GSC cultures. Viability readouts were related to clinical parameters of corresponding patients and whole-transcriptome data. RESULTS: Tumour DNA and RNA sequences revealed strong similarity to corresponding GSCs despite loss of neuronal and immune interactions. In vitro TMZ screening yielded three response categories which significantly correlated with patient survival, therewith providing more specific prediction than the binary MGMT marker. Transcriptome analysis identified 121 genes related to TMZ sensitivity of which 21were validated in external datasets. CONCLUSION: GSCs retain patient-unique hallmark gene expressions despite loss of their natural environment. Drug screening using GSCs predicted patient response to TMZ more specifically than MGMT status, while transcriptome analysis identified potential biomarkers for this response. GSC drug screening therefore provides a tool to improve drug development and precision medicine for glioblastoma.

Novel kinome profiling technology reveals drug treatment is patient and 2D/3D model dependent in glioblastoma.

Glioblastoma is the deadliest brain cancer. One of the main reasons for poor outcome resides in therapy resistance, which adds additional challenges in finding an effective treatment. Small protein kinase inhibitors are molecules that have become widely studied for cancer treatments, including glioblastoma. However, none of these drugs have demonstrated a therapeutic activity or brought more benefit compared to the current standard procedure in clinical trials. Hence, understanding the reasons of the limited efficacy and drug resistance is valuable to develop more effective strategies toward the future. To gain novel insights into the method of action and drug resistance in glioblastoma, we established in parallel two patient-derived glioblastoma 2D and 3D organotypic multicellular spheroids models, and exposed them to a prolonged treatment of three weeks with temozolomide or either the two small protein kinase inhibitors enzastaurin and imatinib. We coupled the phenotypic evidence of cytotoxicity, proliferation, and migration to a novel kinase activity profiling platform (QuantaKinome™) that measured the activities of the intracellular network of kinases affected by the drug treatments. The results revealed a heterogeneous inter-patient phenotypic and molecular response to the different drugs. In general, small differences in kinase activation were observed, suggesting an intrinsic low influence of the drugs to the fundamental cellular processes like proliferation and migration. The pathway analysis indicated that many of the endogenously detected kinases were associated with the ErbB signaling pathway. We showed the intertumoral variability in drug responses, both in terms of efficacy and resistance, indicating the importance of pursuing a more personalized approach. In addition, we observed the influence derived from the application of 2D or 3D models in in vitro studies of kinases involved in the ErbB signaling pathway. We identified in one 3D sample a new resistance mechanism derived from imatinib treatment that results in a more invasive behavior. The present study applied a new approach to detect unique and specific drug effects associated with pathways in in vitro screening of compounds, to foster future drug development strategies for clinical research in glioblastoma.

Quantitative proteomics of small numbers of closely-related cells: Selection of the optimal method for a clinical setting.

Mass spectrometry (MS)-based proteomics profiling has undoubtedly increased the knowledge about cellular processes and functions. However, its applicability for paucicellular sample analyses is currently limited. Although new approaches have been developed for single-cell studies, most of them have not (yet) been standardized and/or require highly specific (often home-built) devices, thereby limiting their broad implementation, particularly in non-specialized settings. To select an optimal MS-oriented proteomics approach applicable in translational research and clinical settings, we assessed 10 different sample preparation procedures in paucicellular samples of closely-related cell types. Particularly, five cell lysis protocols using different chemistries and mechanical forces were combined with two sample clean-up techniques (C18 filter- and SP3-based), followed by tandem mass tag (TMT)-based protein quantification. The evaluation was structured in three phases: first, cell lines from hematopoietic (THP-1) and non-hematopoietic (HT-29) origins were used to test the approaches showing the combination of a urea-based lysis buffer with the SP3 bead-based clean-up system as the best performer. Parameters such as reproducibility, accessibility, spatial distribution, ease of use, processing time and cost were considered. In the second phase, the performance of the method was tested on maturation-related cell populations: three different monocyte subsets from peripheral blood and, for the first time, macrophages/microglia (MAC) from glioblastoma samples, together with T cells from both tissues. The analysis of 50,000 cells down to only 2,500 cells revealed different protein expression profiles associated with the distinct cell populations. Accordingly, a closer relationship was observed between non-classical monocytes and MAC, with the latter showing the co-expression of M1 and M2 macrophage markers, although pro-tumoral and anti-inflammatory proteins were more represented. In the third phase, the results were validated by high-end spectral flow cytometry on paired monocyte/MAC samples to further determine the sensitivity of the MS approach selected. Finally, the feasibility of the method was proven in 194 additional samples corresponding to 38 different cell types, including cells from different tissue origins, cellular lineages, maturation stages and stimuli. In summary, we selected a reproducible, easy-to-implement sample preparation method for MS-based proteomic characterization of paucicellular samples, also applicable in the setting of functionally closely-related cell populations.

3D-Engineered Scaffolds to Study Microtubes and Localization of Epidermal Growth Factor Receptor in Patient-Derived Glioma Cells.

A major obstacle in glioma research is the lack of in vitro models that can retain cellular features of glioma cells in vivo. To overcome this limitation, a 3D-engineered scaffold, fabricated by two-photon polymerization, is developed as a cell culture model system to study patient-derived glioma cells. Scanning electron microscopy, (live cell) confocal microscopy, and immunohistochemistry are employed to assess the 3D model with respect to scaffold colonization, cellular morphology, and epidermal growth factor receptor localization. Both glioma patient-derived cells and established cell lines successfully colonize the scaffolds. Compared to conventional 2D cell cultures, the 3D-engineered scaffolds more closely resemble in vivo glioma cellular features and allow better monitoring of individual cells, cellular protrusions, and intracellular trafficking. Furthermore, less random cell motility and increased stability of cellular networks is observed for cells cultured on the scaffolds. The 3D-engineered glioma scaffolds therefore represent a promising tool for studying brain cancer mechanobiology as well as for drug screening studies.

Drug Repurposing, a Fast-Track Approach to Develop Effective Treatments for Glioblastoma.

Glioblastoma (GBM) remains one of the most difficult tumors to treat. The mean overall survival rate of 15 months and the 5-year survival rate of 5% have not significantly changed for almost 2 decades. Despite progress in understanding the pathophysiology of the disease, no new effective treatments to combine with radiation therapy after surgical tumor debulking have become available since the introduction of temozolomide in 1999. One of the main reasons for this is the scarcity of compounds that cross the blood-brain barrier (BBB) and reach the brain tumor tissue in therapeutically effective concentrations. In this review, we focus on the role of the BBB and its importance in developing brain tumor treatments. Moreover, we discuss drug repurposing, a drug discovery approach to identify potential effective candidates with optimal pharmacokinetic profiles for central nervous system (CNS) penetration and that allows rapid implementation in clinical trials. Additionally, we provide an overview of repurposed candidate drug currently being investigated in GBM at the preclinical and clinical levels. Finally, we highlight the importance of phase 0 trials to confirm tumor drug exposure and we discuss emerging drug delivery technologies as an alternative route to maximize therapeutic efficacy of repurposed candidate drug.

Advancements, Challenges, and Future Directions in Tackling Glioblastoma Resistance to Small Kinase Inhibitors.

Despite clinical intervention, glioblastoma (GBM) remains the deadliest brain tumor in adults. Its incurability is partly related to the establishment of drug resistance, both to standard and novel treatments. In fact, even though small kinase inhibitors have changed the standard clinical practice for several solid cancers, in GBM, they did not fulfill this promise. Drug resistance is thought to arise from the heterogeneity of GBM, which leads the development of several different mechanisms. A better understanding of the evolution and characteristics of drug resistance is of utmost importance to improve the current clinical practice. Therefore, the development of clinically relevant preclinical in vitro models which allow careful dissection of these processes is crucial to gain insights that can be translated to improved therapeutic approaches. In this review, we first discuss the heterogeneity of GBM, which is reflected in the development of several resistance mechanisms. In particular, we address the potential role of drug resistance mechanisms in the failure of small kinase inhibitors in clinical trials. Finally, we discuss strategies to overcome therapy resistance, particularly focusing on the importance of developing in vitro models, and the possible approaches that could be applied to the clinic to manage drug resistance.

Convection Enhanced Delivery of the Oncolytic Adenovirus Delta24-RGD in Patients with Recurrent GBM: A Phase I Clinical Trial Including Correlative Studies.

PURPOSE: Testing safety of Delta24-RGD (DNX-2401), an oncolytic adenovirus, locally delivered by convection enhanced delivery (CED) in tumor and surrounding brain of patients with recurrent glioblastoma. PATIENTS AND METHODS: Dose-escalation phase I study with 3+3 cohorts, dosing 107 to 1 × 1011 viral particles (vp) in 20 patients. Besides clinical parameters, adverse events, and radiologic findings, blood, cerebrospinal fluid (CSF), brain interstitial fluid, and excreta were sampled over time and analyzed for presence of immune response, viral replication, distribution, and shedding. RESULTS: Of 20 enrolled patients, 19 received the oncolytic adenovirus Delta24-RGD, which was found to be safe and feasible. Four patients demonstrated tumor response on MRI, one with complete regression and still alive after 8 years. Most serious adverse events were attributed to increased intracranial pressure caused by either an inflammatory reaction responding to steroid treatment or viral meningitis being transient and self-limiting. Often viral DNA concentrations in CSF increased over time, peaking after 2 to 4 weeks and remaining up to 3 months. Concomitantly Th1- and Th2-associated cytokine levels and numbers of CD3+ T and natural killer cells increased. Posttreatment tumor specimens revealed increased numbers of macrophages and CD4+ and CD8+ T cells. No evidence of viral shedding in excreta was observed. CONCLUSIONS: CED of Delta24-RGD not only in the tumor but also in surrounding brain is safe, induces a local inflammatory reaction, and shows promising clinical responses.

Effects of the IDH1 R132H Mutation on the Energy Metabolism: A Comparison between Tissue and Corresponding Primary Glioma Cell Cultures.

The R132H mutation in the metabolic enzyme isocitrate dehydrogenase 1 (IDH1) is the most important prognostic factor for the survival of glioma patients. Subsequent studies led to the discovery of a panel of enzymes mainly involved in glutamate anaplerosis and aerobic glycolysis that change in abundance as a result of the IDH1 mutation. To further study these changes, appropriate glioma models are required that accurately mimic in vivo metabolism. To investigate how metabolism is affected by in vitro cell culture, we here compared surgically obtained snap-frozen glioma tissues with their corresponding primary glioma cell culture models with a previously developed targeted mass spectrometry proteomic assay. We determined the relative abundance of a panel of metabolic enzymes. Results confirmed increased glutamate use and decreased aerobic glycolysis in resected IDH1 R132H glioma tissue samples. However, these metabolic profiles were not reflected in the paired glioma primary cell cultures. We suggest that culture conditions and tumor microenvironment play a crucial role in maintaining the in vivo metabolic situation in cell culture models. For this reason, new models that more closely resemble the in vivo microenvironment, such as three-dimensional cell co-cultures or organotypic multicellular spheroid models, need to be developed and investigated.

Nonhuman Primate Adenoviruses of the Human Adenovirus B Species Are Potent and Broadly Acting Oncolytic Vector Candidates.

The use of human adenoviruses (hAds) as oncolytic agents has demonstrated considerable potential. However, their efficacy in clinical studies is generally moderate and often varies between patients. This may, in part, be attributable to variable pre-existing neutralizing immunity in patients, which can impact the antitumor efficacy and lead to response heterogeneity. Our aim was to isolate new Ads for the development of oncolytic vectors with low prevalence of neutralizing immunity in the human population. To this end, we isolated a collection of new nonhuman primate (nhp) Ads from stool samples of four great ape species held captive. We elected 12 isolates comprising the broadest genetic variability for further characterization. For three new nhpAds, all classified as the human adenovirus B (HAdV-B) species, no neutralizing activity could be detected when exposed to a preparation of immunoglobulins isolated from a pool of >1,000 donors as a surrogate of population immunity. In addition, the nhpAds of the HAdV-B species showed enhanced oncolytic potency compared to nhpAds of the HAdV-C species as well as to human adenovirus type 5 (HAdV-C5) in vitro when tested in a panel of 29 human cancer cell lines. Next-generation sequencing of the viral genomes revealed higher sequence similarity between hAds and nhpAds of HAdV-B compared to HAdV-C, which might underlie the differences in oncolytic ability. As a proof-of-concept, the Rb-binding domain of the E1A protein of the gorilla-derived HAdV-B nhpAd-lumc007 was deleted, thereby creating a new oncolytic derivative, which demonstrated increased oncolytic potential compared to HAdV-C5. Collectively, our data demonstrate that nhpAds of the HAdV-B species can serve as an alternative for the development of potent oncolytic Ad vectors with limited pre-existing neutralizing immunity in humans.

Generation, characterization, and drug sensitivities of 12 patient-derived IDH1-mutant glioma cell cultures.

BACKGROUND: Mutations of the isocitrate dehydrogenase (IDH) gene occur in over 80% of low-grade gliomas and secondary glioblastomas. Despite considerable efforts, endogenous in vitro IDH-mutated glioma models remain scarce. Availability of these models is key for the development of new therapeutic interventions. METHODS: Cell cultures were established from fresh tumor material and expanded in serum-free culture media. D-2-Hydroxyglutarate levels were determined by mass spectrometry. Genomic and transcriptomic profiling were carried out on the Illumina Novaseq platform, methylation profiling was performed with the Infinium MethylationEpic BeadChip array. Mitochondrial respiration was measured with the Seahorse XF24 Analyzer. Drug screens were performed with an NIH FDA-approved anti-cancer drug set and two IDH-mutant specific inhibitors. RESULTS: A set of twelve patient-derived IDHmt cell cultures was established. We confirmed high concordance in driver mutations, copy numbers and methylation profiles between the tumors and derived cultures. Homozygous deletion of CDKN2A/B was observed in all cultures. IDH-mutant cultures had lower mitochondrial reserve capacity. IDH-mutant specific inhibitors did not affect cell viability or global gene expression. Screening of 107 FDA-approved anti-cancer drugs identified nine compounds with potent activity against IDHmt gliomas, including three compounds with favorable pharmacokinetic characteristics for CNS penetration: teniposide, omacetaxine mepesuccinate, and marizomib. CONCLUSIONS: Our twelve IDH-mutant cell cultures show high similarity to the parental tissues and offer a unique tool to study the biology and drug sensitivities of high-grade IDHmt gliomas in vitro. Our drug screening studies reveal lack of sensitivity to IDHmt inhibitors, but sensitivity to a set of nine available anti-cancer agents.

Transcriptional CDK inhibitors, CYC065 and THZ1 promote Bim-dependent apoptosis in primary and recurrent GBM through cell cycle arrest and Mcl-1 downregulation.

Activation of cyclin-dependent kinases (CDKs) contributes to the uncontrolled proliferation of tumour cells. Genomic alterations that lead to the constitutive activation or overexpression of CDKs can support tumourigenesis including glioblastoma (GBM), the most common and aggressive primary brain tumour in adults. The incurability of GBM highlights the need to discover novel and more effective treatment options. Since CDKs 2, 7 and 9 were found to be overexpressed in GBM, we tested the therapeutic efficacy of two CDK inhibitors (CKIs) (CYC065 and THZ1) in a heterogeneous panel of GBM patient-derived cell lines (PDCLs) cultured as gliomaspheres, as preclinically relevant models. CYC065 and THZ1 treatments suppressed invasion and induced viability loss in the majority of gliomaspheres, irrespective of the mutational background of the GBM cases, but spared primary cortical neurons. Viability loss arose from G2/M cell cycle arrest following treatment and subsequent induction of apoptotic cell death. Treatment efficacies and treatment durations required to induce cell death were associated with proliferation velocities, and apoptosis induction correlated with complete abolishment of Mcl-1 expression, a cell cycle-regulated antiapoptotic Bcl-2 family member. GBM models generally appeared highly dependent on Mcl-1 expression for cell survival, as demonstrated by pharmacological Mcl-1 inhibition or depletion of Mcl-1 expression. Further analyses identified CKI-induced Mcl-1 loss as a prerequisite to establish conditions at which the BH3-only protein Bim can efficiently induce apoptosis, with cellular Bim amounts strongly correlating with treatment efficacy. CKIs reduced proliferation and promoted apoptosis also in chick embryo xenograft models of primary and recurrent GBM. Collectively, these studies highlight the potential of these novel CKIs to suppress growth and induce cell death of patient-derived GBM cultures in vitro and in vivo, warranting further clinical investigation.

The Multifaceted Role of Macrophages in Oncolytic Virotherapy.

One of the cancer hallmarks is immune evasion mediated by the tumour microenvironment (TME). Oncolytic virotherapy is a form of immunotherapy based on the application of oncolytic viruses (OVs) that selectively replicate in and induce the death of tumour cells. Virotherapy confers reciprocal interaction with the host's immune system. The aim of this review is to explore the role of macrophage-mediated responses in oncolytic virotherapy efficacy. The approach was to study current scientific literature in this field in order to give a comprehensive overview of the interactions of OVs and macrophages and their effects on the TME. The innate immune system has a central influence on the TME; tumour-associated macrophages (TAMs) generally have immunosuppressive, tumour-supportive properties. In the context of oncolytic virotherapy, macrophages were initially thought to predominantly contribute to anti-viral responses, impeding viral spread. However, macrophages have now also been found to mediate transport of OV particles and, after TME infiltration, to be subjected to a phenotypic shift that renders them pro-inflammatory and tumour-suppressive. These TAMs can present tumour antigens leading to a systemic, durable, adaptive anti-tumour immune response. After phagocytosis, they can recirculate carrying tissue-derived proteins, which potentially enables the monitoring of OV replication in the TME. Their role in therapeutic efficacy is therefore multifaceted, but based on research applying relevant, immunocompetent tumour models, macrophages are considered to have a central function in anti-cancer activity. These novel insights hold important clinical implications. When optimised, oncolytic virotherapy, mediating multifactorial inhibition of cancer immune evasion, could contribute to improved patient survival.

Transcriptional CDK Inhibitors CYC065 and THZ1 Induce Apoptosis in Glioma Stem Cells Derived from Recurrent GBM.

Glioma stem cells (GSCs) are tumour initiating cells which contribute to treatment resistance, temozolomide (TMZ) chemotherapy and radiotherapy, in glioblastoma (GBM), the most aggressive adult brain tumour. A major contributor to the uncontrolled tumour cell proliferation in GBM is the hyper activation of cyclin-dependent kinases (CDKs). Due to resistance to standard of care, GBMs relapse in almost all patients. Targeting GSCs using transcriptional CDK inhibitors, CYC065 and THZ1 is a potential novel treatment to prevent relapse of the tumour. TCGA-GBM data analysis has shown that the GSC markers, CD133 and CD44 were significantly upregulated in GBM patient tumours compared to non-tumour tissue. CD133 and CD44 stem cell markers were also expressed in gliomaspheres derived from recurrent GBM tumours. Light Sheet Florescence Microscopy (LSFM) further revealed heterogeneous expression of these GSC markers in gliomaspheres. Gliomaspheres from recurrent tumours were highly sensitive to transcriptional CDK inhibitors, CYC065 and THZ1 and underwent apoptosis while being resistant to TMZ. Apoptotic cell death in GSC subpopulations and non-stem tumour cells resulted in sphere disruption. Collectively, our study highlights the potential of these novel CKIs to induce cell death in GSCs from recurrent tumours, warranting further clinical investigation.

Marizomib sensitizes primary glioma cells to apoptosis induced by a latest-generation TRAIL receptor agonist.

Due to the absence of curative treatments for glioblastoma (GBM), we assessed the efficacy of single and combination treatments with a translationally relevant 2nd generation TRAIL-receptor agonist (IZI1551) and the blood-brain barrier (BBB) permeant proteasome inhibitor marizomib in a panel of patient-derived glioblastoma cell lines. These cells were cultured using protocols that maintain the characteristics of primary tumor cells. IZI1551+marizomib combination treatments synergistically induced apoptotic cell death in the majority of cases, both in 2D, as well as in 3D spheroid cultures. In contrast, single-drug treatments largely failed to induce noticeable amounts of cell death. Kinetic analyses suggested that time-shifted drug exposure might further increase responsiveness, with marizomib pre-treatments indeed strongly enhancing cell death. Cell death responses upon the addition of IZI1551 could also be observed in GBM cells that were kept in a medium collected from the basolateral side of a human hCMEC/D3 BBB model that had been exposed to marizomib. Interestingly, the subset of GBM cell lines resistant to IZI1551+marizomib treatments expressed lower surface amounts of TRAIL death receptors, substantially lower amounts of procaspase-8, and increased amounts of cFLIP, suggesting that apoptosis initiation was likely too weak to initiate downstream apoptosis execution. Indeed, experiments in which the mitochondrial apoptosis threshold was lowered by antagonizing Mcl-1 re-established sensitivity to IZI1551+marizomib in otherwise resistant cells. Overall, our study demonstrates a high efficacy of combination treatments with a latest-generation TRAIL receptor agonist and the BBB permeant proteasome inhibitor marizomib in relevant GBM cell models, as well as strategies to further enhance responsiveness and to sensitize subgroups of otherwise resistant GBM cases.

Targeting glioblastoma using a novel peptide specific to a deglycosylated isoform of brevican.

Glioblastoma multiforme (GBM) is the most common and deadliest form of brain tumor and remains amongst the most difficult cancers to treat. Brevican (Bcan), a central nervous system (CNS)-specific extracellular matrix protein, is upregulated in high-grade glioma cells, including GBM. A Bcan isoform lacking most glycosylation, dg-Bcan, is found only in GBM tissues. Here, dg-Bcan is explored as a molecular target for GBM. In this study, we screened a d-peptide library to identify a small 8-amino acid dg-Bcan-Targeting Peptide (BTP) candidate, called BTP-7 that binds dg-Bcan with high affinity and specificity. BTP-7 is preferentially internalized by dg-Bcan-expressing patient-derived GBM cells. To demonstrate GBM targeting, we radiolabeled BTP-7 with 18F, a radioisotope of fluorine, and found increased radiotracer accumulation in intracranial GBM established in mice using positron emission tomography (PET) imaging. dg-Bcan is an attractive molecular target for GBM, and BTP-7 represents a promising lead candidate for further development into novel imaging agents and targeted therapeutics.

Personalizing Oncolytic Virotherapy for Glioblastoma: In Search of Biomarkers for Response.

Oncolytic virus (OV) treatment may offer a new treatment option for the aggressive brain tumor glioblastoma. Clinical trials testing oncolytic viruses in this patient group have shown promising results, with patients achieving impressive long-term clinical responses. However, the number of responders to each OV remains low. This is thought to arise from the large heterogeneity of these tumors, both in terms of molecular make-up and their immune-suppressive microenvironment, leading to variability in responses. An approach that may improve response rates is the personalized utilization of oncolytic viruses against Glioblastoma (GBM), based on specific tumor- or patient-related characteristics. In this review, we discuss potential biomarkers for response to different OVs as well as emerging ex vivo assays that in the future may enable selection of optimal OV for a specific patient and design of stratified clinical OV trials for GBM.

Monocytes carrying GFAP detect glioma, brain metastasis and ischaemic stroke, and predict glioblastoma survival.

Diagnosis and monitoring of primary brain tumours, brain metastasis and acute ischaemic stroke all require invasive, burdensome and costly diagnostics, frequently lacking adequate sensitivity, particularly during disease monitoring. Monocytes are known to migrate to damaged tissues, where they act as tissue macrophages, continuously scavenging, phagocytizing and digesting apoptotic cells and other tissue debris. We hypothesize that upon completion of their tissue-cleaning task, these tissue macrophages might migrate via the lymph system to the bloodstream, where they can be detected and evaluated for their phagolysosomal contents. We discovered a blood monocyte subpopulation carrying the brain-specific glial fibrillary acidic protein in glioma patients and in patients with brain metastasis and evaluated the diagnostic potential of this finding. Blood samples were collected in a cross-sectional study before or during surgery from adult patients with brain lesions suspected of glioma. Together with blood samples from healthy controls, these samples were flowing cytometrically evaluated for intracellular glial fibrillary acidic protein in monocyte subsets. Acute ischaemic stroke patients were tested at multiple time points after onset to evaluate the presence of glial fibrillary acidic protein-carrying monocytes in other forms of brain tissue damage. Clinical data were collected retrospectively. High-grade gliomas (N = 145), brain metastasis (N = 21) and large stroke patients (>100 cm3) (N = 3 versus 6; multiple time points) had significantly increased frequencies of glial fibrillary acidic protein+CD16+ monocytes compared to healthy controls. Based on both a training and validation set, a cut-off value of 0.6% glial fibrillary acidic protein+CD16+ monocytes was established, with 81% sensitivity (95% CI 75-87%) and 85% specificity (95% CI 80-90%) for brain lesion detection. Acute ischaemic strokes of >100 cm3 reached >0.6% of glial fibrillary acidic protein+CD16+ monocytes within the first 2-8 h after hospitalization and subsided within 48 h. Glioblastoma patients with >20% glial fibrillary acidic protein+CD16+ non-classical monocytes had a significantly shorter median overall survival (8.1 versus 12.1 months). Our results and the available literature, support the hypothesis of a tissue-origin of these glial fibrillary acidic protein-carrying monocytes. Blood monocytes carrying glial fibrillary acidic protein have a high sensitivity and specificity for the detection of brain lesions and for glioblastoma patients with a decreased overall survival. Furthermore, their very rapid response to acute tissue damage identifies large areas of ischaemic tissue damage within 8 h after an ischaemic event. These studies are the first to report the clinical applicability for brain tissue damage detection through a minimally invasive diagnostic method, based on blood monocytes and not serum markers, with direct consequences for disease monitoring in future (therapeutic) studies and clinical decision making in glioma and acute ischaemic stroke patients.