Targeting Glioblastoma Stem Cells Research Articles

Approaches in Adult Glioblastoma Treatment: A Systematic Review of Emerging Therapies.

Glioblastoma (GB) is the most common and aggressive primary brain tumor in adults, characterized by complex genetic changes and a poor prognosis. Current standard therapies, including surgery, chemotherapy, and radiotherapy, have limited effectiveness. Emerging therapeutic strategies aim to address the high recurrence rate and improve outcomes by targeting glioblastoma stem cells (GSCs), the blood-brain barrier, and utilizing advanced drug delivery systems. This systematic review followed the Preferred Reporting Items for Systematic Reviews and Meta-Analysis (PRISMA) guidelines. An electronic search was conducted across several databases, including PubMed, Embase, Scopus, Web of Science, and Cochrane, covering studies published from January 2019 to May 2024. The inclusion criteria encompassed primary research studies in English focusing on emerging therapies for treating GB in adults. Eligible studies included experimental and observational studies. Only peer-reviewed journal articles were considered. Exclusion criteria included non-human studies, pediatric studies, non-peer-reviewed articles, systematic reviews, case reports, conference abstracts, and editorials. The search identified 755 articles and, finally, 24 of them met the inclusion criteria. The key findings highlight various promising therapies. Despite advances in treatment approaches, the complexity and heterogeneity of GB necessitate ongoing research to optimize these innovative strategies. The study has limitations that should be considered. The inclusion of only English-language articles may introduce language bias, and the focus on peer-reviewed articles could exclude valuable data from non-peer-reviewed sources. Heterogeneity among studies, particularly in sample sizes and designs, complicates comparison and synthesis, while the reliance on preclinical models limits generalizability to clinical practice. Nonetheless, this review provides a comprehensive overview of the emerging therapies that hold promise for improving patient outcomes in GB treatment.

P02.04.B TARGETING METABOLIC VULNERABILITIES IN GLIOBLASTOMA

Abstract BACKGROUND Glioblastoma is the most common and aggressive primary brain cancer, with a median survival of 16 months. A subset of cells within these tumours, termed glioblastoma stem cells (GSCs), displays high tumourigenic capacity and resistance to conventional therapies, and are responsible for tumour recurrence. Glioblastomas have been initially shown to mainly rely on glycolysis as a source of energy. However, a growing body of literature reports that GSCs are less glycolytic than their differentiated progeny and can thrive on oxidative phosphorylation (OXPHOS) to survive, sustain stemness and fuel tumourigenic potential. Thus, targeting OXPHOS could be a promising strategy to halt energy production and starve GSCs to death. However, under metabolic stress, GSCs can also switch to a more glycolytic phenotype, suggesting a metabolic plasticity in GSCs, and thus adding a layer of complexity to target GSC metabolism. In the current study, we have investigated the impact of compound X (CX), recently shown to impair mitochondrial respiration, on the fate and tumourigenesis of GSCs. MATERIAL AND METHODS Multiple patient-derived and murine GSC models were used to test the impact of CX on GSCs in vitro. The effect of CX on glioblastoma tumors was evaluated using patient-derived xenografts and syngeneic mouse models. RESULTS We have established that CX inhibits GSC proliferation and impairs stemness-related pathways in vitro. CX delays glioblastoma tumourigenesis in vivo. While a drastic decrease in cell proliferation was observed, cell death assays revealed that CX does not induce cell death. This suggests that GSCs gain a slow-cycling phenotype to resist energy depletion. Mechanistically, we have established that CX targets complex I of the mitochondrial respiratory chain, leading to an impairment of OXPHOS. Interestingly, inhibition of OXPHOS by CX was associated with an extensive production of extracellular lactate, indicating that at least, a subset of GSCs gain metabolic adaptability by co-opting glycolysis to survive. Thus, to tackle this metabolic plasticity in GSCs, we adopted a new strategy that involves sequestration of the lactic acid intracellularly via disruption of lactate transporters. Treatment of GSCs with monocarboxylate transporter inhibitors, AZD3965 and syrosingopine, alone did not significantly impact GSC growth and viability. Strikingly, combined CX treatment with either AZD3965 or syrosingopine resulted in high intracellular lactate buildup, increased reactive oxygen species, and led to drastic cell lethality in multiple GSC models. CONCLUSION This study highlights that re-routing the OXPHOS-dependent GSCs to glycolysis offers a vulnerability to sensitize GSCs to cell death. Dual inhibition of OXPHOS and lactate export could be a promising therapeutic approach to deplete malignant GSCs and suppress glioblastoma tumourigenesis.

Exosomal miR-6733-5p mediates cross-talk between glioblastoma stem cells and macrophages and promotes glioblastoma multiform progression synergistically.

Exosomal miRNAs derived from glioblastoma stem cells (GSCs) are important mediators of immunosuppressive microenvironment formation in glioblastoma multiform (GBM), especially in M2-like polarization of tumor-associated macrophages (TAMs). However, the exact mechanisms by which GSCs-derived exosomes (GSCs-exo) facilitate the remodeling of the immunosuppressive microenvironment of GBM have not been elucidated. Transmission electron microscopy (TME) and nanoparticle tracking analysis (NTA) were applied to verify the existence of GSCs-derived exosomes. Sphere formation assays, flow cytometry, and tumor xenograft transplantation assays were performed to identify the exact roles of exosomal miR-6733-5p. Then, the mechanisms of miR-6733-5p and its downstream target gene regulating crosstalk between GSCs cells and M2 macrophages were further investigated. GSCs-derived exosomal miR-6733-5p induce macrophage M2 polarization of TAMs by positively targeting IGF2BP3 to activate the AKT signaling pathway, which further facilitates the self-renewal and stemness of GSCs. GSCs secrete miR-6733-5p-rich exosomes to induce M2-like polarization of macrophages, as well as enhance GSCs stemness and promote malignant behaviors of GBM through IGF2BP3 activated AKT pathway. Targeting GSCs exosomal miR-6733-5p may provide a potential new strategy against GBM.

Abstract 505: Small molecule circadian clock compounds display therapeutic potential in targeting glioblastoma stem cells

Abstract Glioblastoma multiforme (GBM) is the most common primary brain tumor, claiming the lives of roughly 10,000 Americans each year. Despite being the first cancer analyzed through the Cancer Genome Atlas, treatment success remains minimal, resulting average survival time of patients is 15 months following diagnosis and a five-year survival rate is 6.8%. One of the major challenges in treating GBM is the presence of GBM stem cells (GSCs) that are resistant to temozolomide (TMZ) chemotherapy and radiation, which is part of the current standard of care for GBM following maximal surgical resection. We found, however, that GSCs have a unique dependence on core circadian clock proteins, Brain and Muscle ARNTL-Like 1 (BMAL1) and Circadian Locomoter Output Cycles Protein Kaput (CLOCK), which is not observed in differentiated GBM cells or normal neuronal stem cells. Here we explore the use of novel small molecule circadian clock compounds that either lower BMAL1 transcription (REV-ERB agonists) or inhibit BMAL1::CLOCK heterodimer transcriptional activity (Cryptochrome (CRY) stabilizers or Casein Kinase (CK) 1/2 inhibitors) in targeting GSCs in in vitro patient derived cell lines and in in vivo GBM patient-derived xenograft (PDX) models. GSCs display increased sensitivity to clock compounds at single agent and combinations of clock compounds compared to non-cancerous cells, U2OS human osteosarcoma cells, and differentiated GSCs. Additionally, clock compounds are significantly more effective in targeting GSCs than TMZ. The clock compound SHP1705 increased over survival and delayed tumor growth in GBM PDX models. These results highlight the therapeutic potential small molecule circadian clock compounds have against GBM as both a single agent and adjuvant to existing therapies by specifically targeting the GSC population. Citation Format: Priscilla Chan, Lian Wu, Anahit Hovsepyan, Seda Mkhitaryan, Gevorg Karapetyan, Khalid Shah, Hiroaki Wakimoto, Theodore Kamenecka, Laura A. Solt, Jamie Cope, Rex A. Moats, Tsuyoshi Hirota, Jeremy N. Rich, Steve A. Kay. Small molecule circadian clock compounds display therapeutic potential in targeting glioblastoma stem cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 505.

P06.07.A Natural killer cells lyse glioblastoma stem cells and increase their sensitivity to chemotherapy

Abstract Background Glioblastoma is the most common and lethal brain tumor in the adult population and immunotherapy is playing an increasingly central role in the treatment of many cancers. Nevertheless, the search for effective immunotherapeutic approaches for glioblastoma patients continues. In this study, we aimed to explore the therapeutic potential of allogeneic highly activated super-charged natural killer (NK) cells in glioblastoma. Material and Methods Chromium release- and calcein release-based cytotoxicity assays, ELISA, ELISPOT, and multiplex cytokine assays were used to determine NK cell cytotoxicity against glioblastoma stem cells (GSCs) and secretion of cytokines. Cell surface marker expression using flow cytometry and cell growth in vitro and in vivo were measured to determine GSC phenotype. NK cell killing and penetration in 3D were measured using confocal microscopy of GSC tumorospheres. Results Super-charged NK cells efficiently lysed patient-derived GSCs in 2D and 3D models potentially reversing the immunosuppression observed in patients. NK-cells secreted IFN-γ, upregulated GSC surface expression of CD54 and MHC class I and increased sensitivity of GSCs to chemotherapeutic drugs. Co-localization of NK cells with GBM cells in perivascular niches in glioblastoma tissues and their direct contact with GSCs in tumorospheres suggests their ability to infiltrate glioblastoma tumors and target GSCs. Conclusion Allogeneic super-charged NK cells appear to be a potential therapeutic approach for glioblastoma by selectively killing therapy-resistant cancer stem cell population, increasing their immune-related surface markers and enhancing their sensitivity to chemotherapy. Due to GSC heterogeneity and plasticity personalized immunotherapeutic strategies should be developed to effectively target glioblastomas.

Targeting Glioblastoma Stem Cells to Overcome Chemoresistance: An Overview of Current Therapeutic Strategies.

Glioblastoma (GBM) is the most malignant primary brain tumor. The current standard approach in GBM is surgery, followed by treatment with radiation and temozolomide (TMZ); however, GBM is highly resistant to current therapies, and the standard of care has not been revised over the last two decades, indicating an unmet need for new therapies. GBM stem cells (GSCs) are a major cause of chemoresistance due to their ability to confer heterogeneity and tumorigenic capacity. To improve patient outcomes and survival, it is necessary to understand the properties and mechanisms underlying GSC chemoresistance. In this review, we describe the current knowledge on various resistance mechanisms of GBM to therapeutic agents, with a special focus on TMZ, and summarize the recent findings on the intrinsic and extrinsic mechanisms of chemoresistance in GSCs. We also discuss novel therapeutic strategies, including molecular targeting, autophagy inhibition, oncolytic viral therapy, drug repositioning, and targeting of GSC niches, to eliminate GSCs, from basic research findings to ongoing clinical trials. Although the development of effective therapies for GBM is still challenging, this review provides a better understanding of GSCs and offers future directions for successful GBM therapy.

Targeting Glioblastoma Stem Cells: A Review on Biomarkers, Signal Pathways and Targeted Therapy.

Glioblastoma (GBM) remains the most lethal and common primary brain tumor, even after treatment with multiple therapies, such as surgical resection, chemotherapy, and radiation. Although great advances in medical development and improvements in therapeutic methods of GBM have led to a certain extension of the median survival time of patients, prognosis remains poor. The primary cause of its dismal outcomes is the high rate of tumor recurrence, which is closely related to its resistance to standard therapies. During the last decade, glioblastoma stem cells (GSCs) have been successfully isolated from GBM, and it has been demonstrated that these cells are likely to play an indispensable role in the formation, maintenance, and recurrence of GBM tumors, indicating that GSCs are a crucial target for treatment. Herein, we summarize the current knowledge regarding GSCs, their related signaling pathways, resistance mechanisms, crosstalk linking mechanisms, and microenvironment or niche. Subsequently, we present a framework of targeted therapy for GSCs based on direct strategies, including blockade of the pathways necessary to overcome resistance or prevent their function, promotion of GSC differentiation, virotherapy, and indirect strategies, including targeting the perivascular, hypoxic, and immune niches of the GSCs. In summary, targeting GSCs provides a tremendous opportunity for revolutionary approaches to improve the prognosis and therapy of GBM, despite a variety of challenges.

Abstract 1200: Long-duration term TTFields treatment of glioblastoma cells induces cell death

Abstract Glioblastoma is the most aggressive among brain tumors. Radiotherapy and chemotherapy, as adjuvanttreatments after surgical removal of the tumor mass, are often accompanied by undesirable symptoms. Evenwith these available treatment options, patient prognosis is extremely poor. The only approved treatmentoption demonstrating improved life expectancy in the last twenty years has been the application of TumorTreating Fields (TTFields). However, despite different therapeutic actions, tumor relapse occurs inexorably inmost patients. It is essentially unanimously agreed upon that tumor recurrence is due to a small populationof cells named glioblastoma stem cells (GSCs). GSCs represent a restricted cellular subpopulation in the tumormass composed mainly of bulk cells. The main characteristic of GSCs is their tumorigenic capacity even if asmall number of cells are left after surgery. Our working hypothesis is based on evidence that TTFieldsexposure destabilizes the mitotic spindle of rapidly dividing cells to elicit cell death. Thus, TTFields stimulationshould preferentially target bulk cells due to their higher proliferation rates compared to GSCs. Preliminaryexperiments performed using glioblastoma primary cultures obtained from patients' biopsies demonstratedthe presence of two different cell populations. In glioblastoma primary cultures maintained in stem-cellselective medium, more than 90% of glioblastoma cells did not exhibit stemness characteristics. After 72hours of TTFields stimulation, cell death was ascertained in approximately 50% of cells. The remaining viablecells were more than 20% positive for three different stemness markers. Long duration TTFields treatmentexperiments (144 and 288 hours) showed that TTFields at 200 kHz is able to increase the percentage of GSCin glioblastoma cells, yet did exhibit a propensity to reestablish cellular proliferation.In conclusion, long duration TTFields (200 kHz) treatment demonstrated efficacy by inducing cell death anddelaying glioblastoma cell expansion. However, to prevent glioblastoma relapse it is important to combineTTFields with different combination partners that specifically target glioblastoma stem cells. Citation Format: Stefania Castiglione, Matteo Ranucci, Gaetano Cannavale, Francesca Cianci, Ivan Verduci, Michele Mazzanti. Long-duration term TTFields treatment of glioblastoma cells induces cell death [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1200.

Defining phenotypic and functional heterogeneity of glioblastoma stem cells by mass cytometry

Most patients with glioblastoma (GBM) die within 2 years. A major therapeutic goal is to target GBM stem cells (GSCs), a subpopulation of cells that contribute to treatment resistance and recurrence. Since their discovery in 2003, GSCs have been isolated using single-surface markers, such as CD15, CD44, CD133, and α6 integrin. It remains unknown how these single-surface marker–defined GSC populations compare with each other in terms of signaling and function and whether expression of different combinations of these markers is associated with different functional capacity. Using mass cytometry and fresh operating room specimens, we found 15 distinct GSC subpopulations in patients, and they differed in their MEK/ERK, WNT, and AKT pathway activation status. Once in culture, some subpopulations were lost and previously undetectable ones materialized. GSCs that highly expressed all 4 surface markers had the greatest self-renewal capacity, WNT inhibitor sensitivity, and in vivo tumorigenicity. This work highlights the potential signaling and phenotypic diversity of GSCs. Larger patient sample sizes and antibody panels are required to confirm these findings.

Radiation and adjuvant drug-loaded liposomes target glioblastoma stem cells and trigger in-situ immune response.

BackgroundThe radio- and chemo-resistance of glioblastoma stem-like cells (GSCs), together with their innate tumor-initiating aptitude, make this cell population a crucial target for effective therapies. However, targeting GSCs is hardly difficult and complex, due to the presence of the blood–brain barrier (BBB) and the infiltrative nature of GSCs arousing their dispersion within the brain parenchyma.MethodsLiposomes (LIPs), surface-decorated with an Apolipoprotein E-modified peptide (mApoE) to enable BBB crossing, were loaded with doxorubicin (DOXO), as paradigm of cytotoxic drug triggering immunogenic cell death (ICD). Patient-derived xenografts (PDXs) obtained by GSC intracranial injection were treated with mApoE-DOXO-LIPs alone or concomitantly with radiation.ResultsOur results indicated that mApoE, through the engagement of the low-density lipoprotein receptor (LDLR), promotes mApoE-DOXO-LIPs transcytosis across the BBB and confers target specificity towards GSCs. Irradiation enhanced LDLR expression on both BBB and GSCs, thus further promoting LIP diffusion and specificity. When administered in combination with radiations, mApoE-DOXO-LIPs caused a significant reduction of in vivo tumor growth due to GSC apoptosis. GSC apoptosis prompted microglia/macrophage phagocytic activity, together with the activation of the antigen-presenting machinery crucially required for anti-tumor adaptive immune response.ConclusionsOur results advocate for radiotherapy and adjuvant administration of drug-loaded, mApoE-targeted nanovectors as an effective strategy to deliver cytotoxic molecules to GSCs at the surgical tumor margins, the forefront of glioblastoma (GBM) recurrence, circumventing BBB hurdles. DOXO encapsulation proved in situ immune response activation within GBM microenvironment.

Potential Therapeutic Effects of the Neural Stem Cell-Targeting Antibody Nilo1 in Patient-Derived Glioblastoma Stem Cells.

Glioblastoma (GBM) is the most devastating and least treatable brain tumor with median survival <15 months and extremely high recurrence rates. Promising results of immune checkpoint blockade obtained from pre-clinical studies in mice did not translate to clinic, and new strategies are urgently needed, particularly those targeting GBM stem cells (GSCs) that are held responsible for drug resistance and tumor recurrence. Patient-derived GSC cultures are critical for finding effective brain tumor therapies. Here, we investigated the ability of the recently described monoclonal antibody Nilo1 to specifically recognize GSCs isolated from GBM surgical samples. We employed five patient-derived GSC cultures with different stemness marker expression and differentiation potential, able to recapitulate original tumors when xenotransplanted in vivo. To answer whether Nilo1 has any functional effects in patient-derived GSCs lines, we treated the cells with Nilo1 in vitro and analyzed cell proliferation, cell cycle, apoptosis, sphere formation, as well as the expression of stem vs. differentiation markers. All tested GSCs stained positively for Nilo1, and the ability of Nilo1 to recognize GSCs strongly relied on their stem-like phenotype. Our results showed that a subset of patient-derived GSCs were sensitive to Nilo1 treatment. In three GSC lines Nilo1 triggered differentiation accompanied by the induction of p21. Most strikingly, in one GSC line Nilo1 completely abrogated self-renewal and led to Bax-associated apoptosis. Our data suggest that Nilo1 targets a molecule functionally relevant for stemness maintenance and pinpoint Nilo1 as a novel antibody-based therapeutical strategy to be used either alone or in combination with cytotoxic drugs for GSC targeting. Further pre-clinical studies are needed to validate the effectiveness of GSC-specific Nilo1 targeting in vivo.

Advances in histone deacetylase inhibitors in targeting glioblastoma stem cells.

Glioblastoma multiforme (GBM) is a lethal grade IV glioma (WHO classification) and widely prevalent primary brain tumor in adults. GBM tumors harbor cellular heterogeneity with the presence of a small subpopulation of tumor cells, described as GBM cancer stem cells (CSCs) that pose resistance to standard anticancer regimens and eventually mediate aggressive relapse or intractable progressive GBM. Existing conventional anticancer therapies for GBM do not target GBM stem cells and are mostly palliative; therefore, exploration of new strategies to target stem cells of GBM has to be prioritized for the development of effective GBM therapy. Recent developments in the understanding of GBM pathophysiology demonstrated dysregulation of epigenetic mechanisms along with the genetic changes in GBM CSCs. Altered expression/activity of key epigenetic regulators, especially histone deacetylases (HDACs) in GBM stem cells has been associated with poor prognosis; inhibiting the activity of HDACs using histone deacetylase inhibitors (HDACi) has been promising as mono-therapeutic in targeting GBM and in sensitizing GBM stem cells to an existing anticancer regimen. Here, we review the development of pan/selective HDACi as potential anticancer agents in targeting the stem cells of glioblastoma as a mono or combination therapy.

Imidazo[1,2-a]pyridine Derivatives as Aldehyde Dehydrogenase Inhibitors: Novel Chemotypes to Target Glioblastoma Stem Cells.

Glioblastoma multiforme (GBM) is the deadliest form of brain tumor. It is known for its ability to escape the therapeutic options available to date thanks to the presence of a subset of cells endowed with stem-like properties and ability to resist to cytotoxic treatments. As the cytosolic enzyme aldehyde dehydrogenase 1A3 turns out to be overexpressed in these kinds of cells, playing a key role for their vitality, treatments targeting this enzyme may represent a successful strategy to fight GBM. In this work, we describe a novel class of imidazo[1,2-a]pyridine derivatives as aldehyde dehydrogenase 1A3 inhibitors, reporting the evidence of their significance as novel drug candidates for the treatment of GBM. Besides showing an interesting functional profile, in terms of activity against the target enzyme and selectivity toward highly homologous isoenzymes, representative examples of the series also showed a nanomolar to picomolar efficacy against patient-derived GBM stem-like cells, thus proving the concept that targeting aldehyde dehydrogenase might represent a novel and promising way to combat GBM by striking its ability to divide immortally.

Feasibility of Targeting Glioblastoma Stem Cells: From Concept to Clinical Trials.

Glioblastoma is a highly aggressive and invasive brain and Central Nervous System (CNS) tumor. Current treatment options do not prolong overall survival significantly because the disease is highly prone to relapse. Therefore, research to find new therapies is of paramount importance. It has been discovered that glioblastomas contain a population of cells with stem-like properties and that these cells are may be responsible for tumor recurrence. A review of relevant papers and clinical trials in the field was conducted. A PubMed search with related keywords was used to gather the data. For example, "glioblastoma stem cells AND WNT signaling" is an example used to find information on clinical trials using the database ClinicalTrials.gov. Cancer stem cell research has several fundamental issues and uncertainties that should be taken into consideration. Theoretically, a number of treatment options that target glioblastoma stem cells are available for patients. However, only a few of them have obtained promising results in clinical trials. Several strategies are still under investigation. The majority of treatments to target cancer stem cells have failed during clinical trials. Taking into account a number of biases in the field and the number of unsuccessful investigations, the application of the cancer stem cells concept is questionable in clinical settings, at least with respect to glioblastoma.

Targeting Glioblastoma Stem Cells through Disruption of the Circadian Clock

Glioblastomas are highly lethal cancers, containing self-renewing glioblastoma stem cells (GSC). Here, we show that GSCs, differentiated glioblastoma cells (DGC), and nonmalignant brain cultures all displayed robust circadian rhythms, yet GSCs alone displayed exquisite dependence on core clock transcription factors, BMAL1 and CLOCK, for optimal cell growth. Downregulation of BMAL1 or CLOCK in GSCs induced cell-cycle arrest and apoptosis. Chromatin immunoprecipitation revealed that BMAL1 preferentially bound metabolic genes and was associated with active chromatin regions in GSCs compared with neural stem cells. Targeting BMAL1 or CLOCK attenuated mitochondrial metabolic function and reduced expression of tricarboxylic acid cycle enzymes. Small-molecule agonists of two independent BMAL1-CLOCK negative regulators, the cryptochromes and REV-ERBs, downregulated stem cell factors and reduced GSC growth. Combination of cryptochrome and REV-ERB agonists induced synergistic antitumor efficacy. Collectively, these findings show that GSCs co-opt circadian regulators beyond canonical circadian circuitry to promote stemness maintenance and metabolism, offering novel therapeutic paradigms. SIGNIFICANCE: Cancer stem cells are highly malignant tumor-cell populations. We demonstrate that GSCs selectively depend on circadian regulators, with increased binding of the regulators in active chromatin regions promoting tumor metabolism. Supporting clinical relevance, pharmacologic targeting of circadian networks specifically disrupted cancer stem cell growth and self-renewal.This article is highlighted in the In This Issue feature, p. 1469.

Validation of Thiosemicarbazone Compounds as P-Glycoprotein Inhibitors in Human Primary Brain-Blood Barrier and Glioblastoma Stem Cells.

P-glycoprotein (Pgp) is highly expressed on blood-brain barrier (BBB) and glioblastoma (GB) cells, particularly on cancer stem cells (SC). Pgp recognizes a broad spectrum of substrates, limiting the therapeutic efficacy of several chemotherapeutic drugs in eradicating GB SC. Finding effective and safe inhibitors of Pgp that improve drug delivery across the BBB and target GB SC is open to investigation. We previously identified a series of thiosemicarbazone compounds that inhibit Pgp with an EC50 in the nanomolar range, and herein, we investigate the efficacy of three of them in bypassing Pgp-mediated drug efflux in primary human BBB and GB cells. At 10 nM, the compounds were not cytotoxic for the brain microvascular endothelial hCMEC/D3 cell line, but they markedly enhanced the permeability of the Pgp-substrate doxorubicin through the BBB. Thiosemicarbazone derivatives increased doxorubicin uptake in GB, with greater effects in the Pgp-rich SC clones than in the differentiated clones derived from the same tumor. All compounds increased intratumor doxorubicin accumulation and consequent toxicity in GB growing under competent BBB, producing significant killing of GB SC. The compounds crossed the BBB monolayer. The most stable derivative, 10a, had a half-life in serum of 4.2 h. The coadministration of doxorubicin plus 10a significantly reduced the growth of orthotopic GB-SC xenografts, without eliciting toxic side effects. Our work suggests that the thiosemicarbazone compounds are able to transform doxorubicin, a prototype BBB-impermeable drug, into a BBB-permeable drug. Bypassing Pgp-mediated drug efflux in both BBB and GB SC, thiosemicarbazones might increase the success of chemotherapy in targeting GB SC, which represent the most aggressive and difficult components to eradicate.

Targeting Glioblastoma Stem Cells with 2-Deoxy-D-Glucose (2-DG) Potentiates Radiation-Induced Unfolded Protein Response (UPR)

Glioblastoma (GBM) is the most common and aggressive primary brain tumor in adults, and despite optimized treatment options, median survival remains dismal. Contemporary evidence suggests disease recurrence results from expansion of a robustly radioresistant subset of GBM progenitor cells, termed GBM stem cells (GSCs). In this study, we utilized transmission electron microscopy to uncover ultrastructural effects on patient-derived GSC lines exposed to supratherapeutic radiotherapy levels. Elevated autophagosome formation and increased endoplasmic reticulum (ER) internal diameter, a surrogate for ER stress and activation of unfolded protein response (UPR), was uncovered. These observations were confirmed via protein expression through Western blot. Upon interrogating genomic data from an open-access GBM patient database, overexpression of UPR-related chaperone protein genes was inversely correlated with patient survival. This indicated controlled UPR may play a role in promoting radioresistance. To determine if potentiating UPR further can induce apoptosis, we exposed GSCs to radiation with an ER stress-inducing drug, 2-deoxy-D-glucose (2-DG), and found dose-dependent decreases in viability and increased apoptotic marker expression. Taken together, our results indicate GSC radioresistance is, in part, achieved by overexpression and overactivation of ER stress-related pathways, and this effect can be overcome via potentiation of UPR, leading to loss of GSC viability.

Abstract A068: Targeting glioblastoma stem cells through a MET inhibitor

Abstract Background: Glioblastoma (GBM) is the most common and aggressive human brain cancer with high relapse rate, lacking any targeted therapy. The current therapy consisting of surgery in combination with radiotherapy and the DNA-alkylating agent temozolomide has led to median survival increase. Nevertheless, many patients, after early treatment response, experience drug resistance and tumor progression. Recent advances identified cancer stem cell subpopulation as the cause of treatment failure, suggesting that the direct targeting of this cell subset may represent an innovative approach to improve the therapy efficacy. Based on studies indicating the role of the tyrosine kinase receptor MET in GBM stem cells, we examined the effects of the rational combination of radiotherapy with a MET inhibitor against stem-like cells from a human GBM cell line. Methods: Stem-like cells from U87MG cell line were sorted using PKH-67-labeling method for isolating slow-dividing cells (PKH67+), and analyzed for cell proliferation index by WST assay, clonogenic potential, and expression of stemness-related transcription factors by RT-PCR. Effects by the MET inhibitor alone and in combination with radiotherapy (IR) were evaluated on cell proliferation by Trypan Blue exclusion test, on apoptosis, cell cycle progression, GFAP protein expression by flow cytometry and on neurosphere-forming efficiency. The mechanisms underlying the effects induced by the combined treatment were dissected by biochemical and RT-PCR assays. Results: The PKH-67+ cells recapitulated the functional properties of stem-like cells, as slower cell proliferation index, higher clonogenic capacity, and expression of stemness-transcription factors than the negative counterpart. They also displayed a marked radioresistance dependent on MET signalling activation and characterized by the enrichment in stem-like cells as indicated by the upregulation of stemness-related transcription factor expression Nanog and Sox2, and enhanced susceptibility to MET inhibitor. The single-agent therapy with the inhibitor induced cell proliferation inhibition with an early cell cycle arrest in G2/M phase and a late cytotoxic effect. The drug alone negatively affected the neurosphere-forming efficiency and the clonogenic capacity. Following IR, the daily treatment with the inhibitor of MET significantly caused time-dependent cell proliferation inhibition, associated with an increased percentage of apoptotic cells and cells positive for GFAP, a glial differentiation marker, as compared to single-agent treatments. The biochemical analysis confirmed that MET inhibition remarkably impaired the radioresistance, and parallelly reduced the content of stem-like cells, as indicated either by the negative regulation of Nanog and Sox2 genes or the positive regulation of GFAP. Conclusions: Our results provide evidence that targeting MET with a specific inhibitor might interfere with MET-mediated radiotherapy resistance, thus sensitizing the GBM-stem-like cells to radiation, depleting the pool, and parallelly inducing their reprogramming towards more differentiated subtype. Citation Format: Anna Rossini, Evelyn Oliva Savoia, Eleonora Cicoria, Lorenzo Castagnoli, Serenella Pupa, Filippo de Braud, Massimo Di Nicola. Targeting glioblastoma stem cells through a MET inhibitor [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr A068.

Targeting glioblastoma stem-cells: a recurrent challenge in neurooncology

Glioblastoma (GBM) is the most common and most aggressive primary brain tumor in adults. To date, this tumor remains rapidly fatal despite treatment, median overall survival not exceeding 15 months from diagnosis (1).

Targeting glioblastoma stem-cells: a recurrent challenge in neuro-oncology

Targeting glioblastoma stem-cells: a recurrent challenge in neuro-oncology