Glioblastoma Stem-like Cells Research Articles

Epigenetic modulator inhibition overcomes temozolomide chemoresistance and antagonizes tumor recurrence of glioblastoma.

Glioblastoma multiforme (GBM) heterogeneity causes a greater number of deaths than any other brain tumor, despite the availability of alkylating chemotherapy. GBM stem-like cells (GSCs) contribute to GBM complexity and chemoresistance, but it remains challenging to identify and target GSCs or factors that control their activity. Here, we identified a specific GSC subset and show that activity of these cells is positively regulated by stabilization of methyl CpG binding domain 3 (MBD3) protein. MBD3 binds to CK1A and to BTRCP E3 ubiquitin ligase, triggering MBD3 degradation, suggesting that modulating this circuit could antagonize GBM recurrence. Accordingly, xenograft mice treated with the CK1A activator pyrvinium pamoate (Pyr-Pam) showed enhanced MBD3 degradation in cells expressing high levels of O6-methylguanine-DNA methyltransferase (MGMT) and in GSCs, overcoming temozolomide chemoresistance. Pyr-Pam blocked recruitment of MBD3 and the repressive nucleosome remodeling and deacetylase (NuRD) complex to neurogenesis-associated gene loci and increased acetyl-histone H3 activity and GSC differentiation. We conclude that CK1A/BTRCP/MBD3/NuRD signaling modulates GSC activation and malignancy, and that targeting this signaling could suppress GSC proliferation and GBM recurrence.

RBPJ contributes to the malignancy of glioblastoma and induction of proneural-mesenchymal transition via IL-6-STAT3 pathway.

Notch signaling plays a pivotal role in many cancers, including glioblastoma (GBM). Recombination signal binding protein for immunoglobulin kappa J region (RBPJ) is a key transcription factor of the Notch signaling pathway. Here, we interrogated the function of RBPJ in GBM. Firstly, RBPJ expression of GBM samples was examined. Then, we knocked down RBPJ expression in 2 GBM cell lines (U251 and T98) and 4 glioblastoma (GBM) stem‐like cell lines derived from surgical samples of GBM (KGS01, KGS07, KGS10 and KGS15) to investigate the effect on cell proliferation, invasion, stemness, and tumor formation ability. Expression of possible downstream targets of RBPJ was also assessed. RBPJ was overexpressed in the GBM samples, downregulation of RBPJ reduced cell proliferation and the invasion ability of U251 and T98 cells and cell proliferation ability and stemness of glioblastoma stem‐like cells (GSC) lines. These were accompanied by reduced IL‐6 expression, reduced activation of STAT3, and inhibited proneural‐mesenchymal transition (PMT). Tumor formation and PMT were also impaired by RBPJ knockdown in vivo. In conclusion, RBPJ promotes cell proliferation, invasion, stemness, and tumor initiation ability in GBM cells through enhanced activation of IL‐6‐STAT3 pathway and PMT, inhibition of RBPJ may constitute a prospective treatment for GBM.

Temozolomide-Induced RNA Interactome Uncovers Novel LncRNA Regulatory Loops in Glioblastoma.

Simple SummaryGlioblastoma (GBM) is the most aggressive brain tumor and most resistant to therapy. The identification of novel predictive biomarkers or targets to counteract chemoresistance, requires a better understanding of the GBM primary response to therapy. The aim of our study was to assess the molecular response of GBM to the standard of care chemotherapy by temozolomide (TMZ). We established a comprehensive map of gene expression changes after treatment and discovered that GBM cells elicit a coordinated gene expression program after chemotherapy that differs between sensitive and resistant cells. We found that a novel class of genes expressed as long non-coding RNAs (lncRNAs) is involved in gene regulatory circuits in GBM and could represent novel markers of GBM patient prognosis. By shedding light on the involvement of the non-coding genome in GBM, our results may provide new mechanistic insight on lncRNAs and their importance in chemoresistance. Resistance to chemotherapy by temozolomide (TMZ) is a major cause of glioblastoma (GBM) recurrence. So far, attempts to characterize factors that contribute to TMZ sensitivity have largely focused on protein-coding genes, and failed to provide effective therapeutic targets. Long noncoding RNAs (lncRNAs) are essential regulators of epigenetic-driven cell diversification, yet, their contribution to the transcriptional response to drugs is less understood. Here, we performed RNA-seq and small RNA-seq to provide a comprehensive map of transcriptome regulation upon TMZ in patient-derived GBM stem-like cells displaying different drug sensitivity. In a search for regulatory mechanisms, we integrated thousands of molecular associations stored in public databases to generate a background “RNA interactome”. Our systems-level analysis uncovered a coordinated program of TMZ response reflected by regulatory circuits that involve transcription factors, mRNAs, miRNAs, and lncRNAs. We discovered 22 lncRNAs involved in regulatory loops and/or with functional relevance in drug response and prognostic value in gliomas. Thus, the investigation of TMZ-induced gene networks highlights novel RNA-based predictors of chemosensitivity in GBM. The computational modeling used to identify regulatory circuits underlying drug response and prioritizing gene candidates for functional validation is applicable to other datasets.

Biological Characterization and Therapeutics for Subscalp Recurrent in Intracranial Glioblastoma.

PurposeGliomas are common intracranial tumors, of which 70% are malignant gliomas. Glioblastoma multiforme (GBM) is the most aggressive tumor, and patients with GBM have a median survival time of only 9–12 months; extracranial recurrence of GBM is very rare. A therapeutic strategy for this kind of recurrent tumor is lacking.Materials and MethodsWe present a case of a patient with extracranial recurrence of subscalp GBM. The subscalp tumor was resected and xenotransplanted into BALB/C nude mice. Then, glioma cells were isolated from the xenograft models and passaged in vitro. HE staining, immunohistochemistry, CCK-8 assays, karyotypic analysis, short tandem repeat STR analysis and flow cytometry were used to analyze the biological characteristics and malignant phenotype of these established cells. The cells and xenografts were then used as preclinical models to evaluate the antitumor efficacy of oncolytic herpes simplex virus 1 (oHSV-1).ResultsThe isolated cells, which were named BT-01, were positive for Nestin and GFAP. The main characteristics of BT-01 cells were that they harbored glioblastoma stem-like cells (GSCs) and that they possessed highly aggressive migration capacities compared with the existing cell lines U87-MG and U251-MG. Moreover, BT-01 cells tolerated the chemotherapeutic drug temozolomide. Our study showed that oHSV-1 could replicate in and repress the growth of BT-01 cells and significantly inhibit tumor growth in xenograft models.ConclusionTaken together, our results showed that a new recurrent glioblastoma cell line was established, which can be useful for research on recurrent glioblastoma. We provided a reliable preclinical model to evaluate the antitumor efficacy of oHSV-1 in vivo and a promising therapy for recurrent GBM.

Efficient Multi-Allelic Genome Editing of Primary Cell Cultures via CRISPR-Cas9 Ribonucleoprotein Nucleofection.

CRISPR-Cas9-based technologies have revolutionized experimental manipulation of mammalian genomes. However, limitations regarding the delivery and efficacy of these technologies restrict their application in primary cells. This article describes a protocol for penetrant, reproducible, and fast CRISPR-Cas9 genome editing in cell cultures derived from primary cells. The protocol employs transient nucleofection of ribonucleoprotein complexes composed of chemically synthesized 2'-O-methyl-3'phosphorothioate-modified single guide RNAs (sgRNAs) and purified Cas9 protein. It can be used both for targeted insertion-deletion mutation (indel) formation at up to >90% efficiency (via use of a single sgRNA) and for targeted deletion of genomic regions (via combined use of multiple sgRNAs). This article provides examples of the nucleofection buffer and programs that are optimal for patient-derived glioblastoma (GBM) stem-like cells (GSCs) and human neural stem/progenitor cells (NSCs), but the protocol can be readily applied to other primary cell cultures by modifying the nucleofection conditions. In summary, this is a relatively simple method that can be used for highly efficient and fast gene knockout, as well as for targeted genomic deletions, even in hyperdiploid cells such as many cancer stem-like cells. © 2020 Wiley Periodicals LLC Basic Protocol: Cas9:sgRNA ribonucleoprotein nucleofection for insertion-deletion (indel) mutation and genomic deletion generation in primary cell cultures.

Decreased Equilibrative Nucleoside Transporter 1 (ENT1) Activity Contributes to the High Extracellular Adenosine Levels in Mesenchymal Glioblastoma Stem-Like Cells.

Glioblastoma multiforme is one of the most malignant types of cancer. This is mainly due to a cell subpopulation with an extremely aggressive potential, called glioblastoma stem-like cells (GSCs). These cells produce high levels of extracellular adenosine which has been associated with increased chemoresistance, migration, and invasion in glioblastoma. In this study, we attempted to elucidate the mechanisms that control extracellular adenosine levels in GSC subtypes. By using primary and U87MG-derived GSCs, we associated increased extracellular adenosine with the mesenchymal phenotype. [3H]-adenosine uptake occurred mainly through the equilibrative nucleoside transporters (ENTs) in GSCs, but mesenchymal GSCs have lower expression and ENT1-mediated uptake activity than proneural GSCs. By analyzing expression and enzymatic activity, we determined that ecto-5′-nucleotidase (CD73) is predominantly expressed in proneural GSCs, driving AMPase activity. While in mesenchymal GSCs, both CD73 and Prostatic Acid Phosphatase (PAP) contribute to the AMP (adenosine monophosphate) hydrolysis. We did not observe significant differences between the expression of proteins involved in the metabolization of adenosine among the GCSs subtypes. In conclusion, the lower expression and activity of the ENT1 transporter in mesenchymal GSCs contributes to the high level of extracellular adenosine that these GSCs present.

Anarchy or Respect the Hierarchy? The Complexity of Glioblastoma.

Glioma stem-like cells (GSC) in glioblastoma (GBM) structure tumor cells into a hierarchical organization and are postulated to be recalcitrant to conventional treatments, resulting in fatal relapse of the disease. A better understanding of these cells would be essential for meaningful and lasting treatments. In this issue of Cancer Research, Virolle and colleagues report a fascinating phenotype whereby the extracellular signal-regulated kinase (ERK) pathway regulates a mechanism of dedifferentiation of GBM cells into a stem-like state expressing markers of pluripotency through an EGFR-ERK-EGR1-dependent axis. This aptly termed "toggle switch" may contribute to maintenance of GSCs, promote intratumor heterogeneity, and potentially provide innovative treatment options.See related article by Almairac et al., p. 3236.

Abstract 4956: PrPc depletion in glioblastoma patient derived xenograft cells by CRISPR/Cas9

Abstract Glioblastoma (GBM), the most common primary malignant brain tumor, exhibits a highly proliferative cell subpopulation called Glioblastoma stem-like cells (GSCs), presumed to contribute to the aggressive behavior of GBM. Our group has shown that the Prion Protein (PrPC), a cell surface glycoprotein and regulator of several stem cell functions, is enriched in GSCs and co-expresses with conventional GSCs markers. PrPC has been proposed as a scaffold protein, able to govern the assembly of multi-component complexes in the cell membrane. Integrin α6 (ITGA6), a cell surface laminin receptor, is enriched in GSCs as well, and exhibits relevant effects on GSCs biology, related to self-renewal and migration. To investigate a potential association between PrPC and ITGA6, we will perform loss-of-function experiments in GSCs via siRNA and CRISPR/Cas9 to characterize these cell surface molecules expression and their effect on GSCs proliferation, migration and invasion. Preliminary results show that depletion of PrPC perturbates ITGA6 labeling on plasma membrane of GSC. Thus far, cells from patient-derived xenografts (GBM33-PDX2) were transfected a construct for PrPC knockout by CRISPR/Cas9 via Nucleofector Technology. We found around 60% GFP-positive cells via flow cytometry for the CRISPR pool and we are currently isolating clones to confirm PrPC expression. Remarkably, the colonies exhibited distinct morphologies such as neural-like, epithelial-like and stem cell-like, indicating the heterogenous nature of this tumor. Western blotting assays will be performed to assess PrPC and ITGA6 expression in both commercial cell line U87 and GBM33-PDX2. The characterization of PrPC-ITGA6 association might provide insight into GSCs behavior and new anti-GBM therapeutic strategies. Citation Format: Maria Isabel Melo Escobar, Barbara Paranhos Coelho, Mariana Brandão Prado, Rebeca Iglesia, Marilene Hohmuth Lopes. PrPc depletion in glioblastoma patient derived xenograft cells by CRISPR/Cas9 [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4956.

Abstract 1929: A confirmatory study to probe the reproducibility of cancer cell lines as lab tools to identify molecular subtype-specific therapies and therapeutic targets against glioblastoma stem-like cells

Abstract Objective: Evidence that some high-impact biomedical results cannot be repeated has stimulated interest in development of standardization and normalization tools. Cancer cell lines are the workhorse of preclinical cancer research and their ability as test-matrix to prove reproducible results has been challenged. Stratifying tumors according to their DNA-methylation and transcriptome has revolutionized neuropathology and raises hopes to develop personalized management options for brain cancer patients. Here, we probe the reproducibility of glioblastoma stem-like cell lines (GSCs) to function as a study tool for the identification of tumor-subtype-specific therapeutic targets and therapies. Methods: A collection of phenotypically diverse GSCs was assembled. In vitro aging was enforced by controlled passaging of n=30 passages and comparative analysis of aging markers before and at the end of the experiment. High-resolution (at least n=5 different concentrations of the substances), medium-throughput (231 clinical-relevant drugs) pharmacology was performed using automation-assistance. Therapy response is presented by quantification of IC50 and AUC in terms of inhibition of cellular growth. Quantification of gene expression was performed using whole transcriptome RNA sequencing and global DNA-methylation was characterized using EPIC BeadChip. In silico prediction of drug response was performed by correlating the transcriptome with gene expression/drug-response data retrieved from publically-available sources. Results: Classification of cell models into transcriptional tumor subtypes was feasible but models show limited difference in DNA-methylation. Reproducibly of drug-response was limited with significant alterations observed in every 8th of the tested compounds. Our comparative results provide a large and technical most accurate alignment of in silico drug screen of GSCs to its physical acquired counterpart. Conclusions: GSCs are a practical study tool to perform throughput pharmacology testing in molecular subtype-specific manner. Unequivocal association of cell models into established molecular subtypes as identified in clinical specimens is partly feasible. Meanwhile our project is ongoing, we hypothesize the generation of a multi OMICS/chemo responsome compendium is an valuable approach to increase the robustness of cell line-guided studies aiming to discover therapeutic targets and therapeutics. Citation Format: Ann-Christin Nickel, Daniel Picard, Gonzalo Torga, Hans-Jakob Steiger, Daniel Hänggi, Maria Stella Carro, Marc Remke, Ulf Dietrich Kahlert. A confirmatory study to probe the reproducibility of cancer cell lines as lab tools to identify molecular subtype-specific therapies and therapeutic targets against glioblastoma stem-like cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1929.

Abstract 3907: Invasive properties of GSCs with known IDH1 status into human cerebral organoids

Abstract Glioblastomas (GBM) are malignant Grade IV tumors whose prognosis for patients remains dismal, with median survival of 14.6 months after the standard therapy. GBMs have a high proliferative index and are highly invasive to normal brain tissue. GBMs characteristically exhibit areas of necrosis and recruitment of abnormal blood vessels. The most common form of GBMs are primary GBMs (~95%), with no prior clinical history and have poor clinical outcome. Secondary GBMs arise from lower-grade gliomas and are associated with IDH1 mutation (R132H/C/G/S). Methylation profiling identified a subtype of GBMs called glioma-CpG island methylator phenotype (G-CIMP+ve) GBMs, and this subtype yields significant survival benefit compared to non-G-CIMP+ve GBMs and is mostly associated with IDH1 mutant GBMs. GBM biology is traditionally investigated by utilizing patient-derived tumor cells (e.g GBM stem-like cells “GSCs”) in vitro and in vivo as orthotopic xenografts in immunocompromised mouse models. However, these systems lack normal human neural tissue, and therefore the contribution of the normal human neural microenvironment is unable to be studied using these systems. This shortcoming can be addressed by utilizing human cerebral organoids (COs). Cerebral organoids are derived from induced pluripotent stem cells and can serve as in vitro model systems to recapitulate the human brain microenvironment where multiple neural cell types are represented. A recent study showed that GSCs invade and proliferate in human cerebral organoids, and these tumors phenocopy parental tumors. Here, we hypothesized that the invasiveness of GSCs within COs is dictated by IDH1 status, where IDH1 wild-type are more aggressive than IDH1 mutant GSCs. To test our hypothesis, we utilized 45 day-old COs and three GSCs lines (2 IDH1 wild-type and 1 IDH1 mutant) for in vitro invasion and proliferation assays. These GSCs stably express either green or red fluorescent proteins and can be used as surrogates to identify GSCs within COs. Upon co-incubation for various time points, these GSC bearing COs were fixed, embedded, sectioned, and imaged with a confocal microscope. Our preliminary results show that GSCs with wild-type IDH1 appear to be more aggressive within COs. With further research studies, the use of COs will elucidate the contribution of the normal brain microenvironment to the growth and invasiveness of GBMs. Citation Format: Maxime Munyeshyaka, Sanjay Singh, Joy Gumin, Jing Yang, Daniel Ledbetter, Frederick Lang. Invasive properties of GSCs with known IDH1 status into human cerebral organoids [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3907.

The different role of YKL-40 in glioblastoma is a function of MGMT promoter methylation status

Inter- and intratumoral heterogeneity is a hallmark of glioblastoma (GBM) that facilitates recurrence, treatment resistance, and worse prognosis. O6-methylguanine-DNA methyltransferase (MGMT) promoter methylation is a significant prognostic marker for Temozolomide (TMZ) resistance in GBM patients. YKL-40 is a molecular marker for the mesenchymal subtype of GBMs and is responsible for TMZ resistance. However, underlying mechanisms by which MGMT epigenetics impacts patient outcomes and the function of YKL-40 are not fully determined. Herein, we performed in vitro and in vivo experiments, six human IDH1/2 wild-type glioblastoma stem-like cells (GSCs) were established and studied to further determine a potential interaction of YKL-40 and MGMT promoter methylation. We demonstrated that YKL-40 functioned differently in human IDH1/2 wild-type GSCs. In MGMT promoter-methylated (MGMT-m) GSCs, it acted as a tumor suppressor gene. On the other hand, in MGMT promoter-unmethylated (MGMT-um) GSCs, it promoted tumorigenesis. Notably, the reason that YKL-40 played different roles in GSCs could not be interpreted by the molecular classification of each GSCs, but is a function of MGMT promoter methylation status and involves the RAS–MEK–ERK pathway. YKL-40 mediated TMZ sensitivity by activating DNA damage responses (DDRs) in MGMT-m GSCs, and it mediated resistance to TMZ by inhibiting DDRs in MGMT-um GSCs. Our report demonstrated that MGMT promoter methylation status might influence a gene’s function in human cancer. Moreover, our data also highlight the point that gene function should be investigated not only according to the molecular tumor classification, but also the epigenetic signature.

A simple and highly efficient method for multi-allelic CRISPR-Cas9 editing in primary cell cultures.

BackgroundCRISPR‐Cas9‐based technologies have revolutionized experimental manipulation of mammalian genomes. None‐the‐less, limitations of the delivery and efficacy of these technologies restrict their application in primary cells.AimsTo create an optimized protocol for penetrant, reproducible, and fast targeted genome editing in cell cultures derived from primary cells, using patient‐derived glioblastoma stem‐like cells (GSCs) and human neural stem/progenitor cells (NSCs) for proof‐of‐concept experiments.Methods and resultsWe employed transient nucleofection of Cas9:sgRNA ribonucleoprotein complexes composed of chemically synthesized 2′‐O‐methyl 3′phosphorothioate‐modified sgRNAs and purified Cas9 protein. Insertion‐deletion mutation (indel) frequency and size distribution were measured via computational deconvolution of Sanger sequencing trace data. We found that this optimized technique routinely allows for >90% indel formation in only 3 days, without the need to create clonal lines for simple loss‐of‐function experiments. Using Western blotting, we observed near‐total protein loss of target genes in cell pools. Additionally, we found that this approach allows for the creation of targeted genomic deletions. Furthermore, by using RNA‐seq in edited NSCs to assess gene expression changes resulting from knockout of tumor suppressors commonly altered in glioblastoma, we also demonstrated the utility of this method for quickly creating a series of gene knockouts that allow for the study of oncogenic activities.ConclusionOur data suggest that this relatively simple method can be used for highly efficient and fast gene knockout, as well as for targeted genomic deletions, even in hyperdiploid cells (such as GSCs). This represents an extremely useful tool for the cancer research community when wishing to inactivate not only coding genes, but also non‐coding RNAs, UTRs, enhancers, and promoters. This method can be readily applied to diverse cell types by varying the nucleofection conditions.

Different Mechanisms Underlie the Metabolic Response of GBM Stem-Like Cells to Ionizing Radiation: Biological and MRS Studies on Effects of Photons and Carbon Ions.

Glioblastoma multiforme (GBM) is a malignant primary brain tumor with very poor prognosis, high recurrence rate, and failure of chemo-radiotherapy, mainly due to a small fraction of cells with stem-like properties (GSCs). To study the mechanisms of GSCs resistance to radiation, two GSC lines, named line #1 and line #83, with different metabolic patterns and clinical outcome, were irradiated with photon beams and carbon ions and assessed by 1H Magnetic Resonance Spectroscopy (MRS). Both irradiation modalities induced early cytotoxic effects in line #1 with small effects on cell cycle, whereas a proliferative G2/M cytostatic block was observed in line #83. MR spectroscopy signals from mobile lipids (ML) increased in spectra of line #1 after photon and C-ion irradiation with effects on lipid unsaturation level, whereas no effects were detected in line #83 spectra. Gamma-Aminobutyric Acid (GABA), glutamic acid (glu) and Phosphocreatine (pCr) signals showed a significant variation only for line #1 after carbon ion irradiation. Glucose (glc) level and lactate (Lac) extrusion behaved differently in the two lines. Our findings suggest that the differences in irradiation response of GSCs #1 and #83 lines are likely attributable to their different metabolic fingerprint rather than to the different radiation types.

Single-cell lineage analysis reveals genetic and epigenetic interplay in glioblastoma drug resistance

BackgroundTumors can evolve and adapt to therapeutic pressure by acquiring genetic and epigenetic alterations that may be transient or stable. A precise understanding of how such events contribute to intratumoral heterogeneity, dynamic subpopulations, and overall tumor fitness will require experimental approaches to prospectively label, track, and characterize resistant or otherwise adaptive populations at the single-cell level. In glioblastoma, poor efficacy of receptor tyrosine kinase (RTK) therapies has been alternatively ascribed to genetic heterogeneity or to epigenetic transitions that circumvent signaling blockade.ResultsWe combine cell lineage barcoding and single-cell transcriptomics to trace the emergence of drug resistance in stem-like glioblastoma cells treated with RTK inhibitors. Whereas a broad variety of barcoded lineages adopt a Notch-dependent persister phenotype that sustains them through early drug exposure, rare subclones acquire genetic changes that enable their rapid outgrowth over time. Single-cell analyses reveal that these genetic subclones gain copy number amplifications of the insulin receptor substrate-1 and substrate-2 (IRS1 or IRS2) loci, which activate insulin and AKT signaling programs. Persister-like cells and genomic amplifications of IRS2 and other loci are evident in primary glioblastomas and may underlie the inefficacy of targeted therapies in this disease.ConclusionsA method for combined lineage tracing and scRNA-seq reveals the interplay between complementary genetic and epigenetic mechanisms of resistance in a heterogeneous glioblastoma tumor model.

Rapid Processing and Drug Evaluation in Glioblastoma Patient-Derived Organoid Models with 4D Bioprinted Arrays

SummaryGlioblastoma is the most common and deadly primary brain malignancy. Despite advances in precision medicine oncology (PMO) allowing the identification of molecular vulnerabilities in glioblastoma, treatment options remain limited, and molecular assays guided by genomic and expression profiling to inform patient enrollment in life-saving trials are lacking. Here, we generate four-dimensional (4D) cell-culture arrays for rapid assessment of drug responses in glioblastoma patient-derived models. The arrays are 3D printed with thermo-responsive shape memory polymer (SMP). Upon heating, the SMP arrays self-transform in time from 3D cell-culture inserts into histological cassettes. We assess the utility of these arrays with glioblastoma cells, gliospheres, and patient derived organoid-like (PDO) models and demonstrate their use with glioblastoma PDOs for assessing drug sensitivity, on-target activity, and synergy in drug combinations. When including genomic and drug testing assays, this platform is poised to offer rapid functional drug assessments for future selection of therapies in PMO.

Molecular analyses of glioblastoma stem-like cells and glioblastoma tissue.

Glioblastoma is a common, malignant brain tumor whose disease incidence increases with age. Glioblastoma stem-like cells (GSCs) are thought to contribute to cancer therapy resistance and to be responsible for tumor initiation, maintenance, and recurrence. This study utilizes both SNP array and gene expression profiling to better understand GSCs and their relation to malignant disease. Peripheral blood and primary glioblastoma tumor tissue were obtained from patients, the latter of which was used to generate GSCs as well as a CD133pos./CD15pos. subpopulation. The stem cell features of GSCs were confirmed via the immunofluorescent expression of Nestin, SOX2, and CD133. Both tumor tissue and the isolated primary cells shared unique abnormal genomic characteristics, including a gain of chromosome 7 as well as either a partial or complete loss of chromosome 10. Individual genomic differences were also observed, including the loss of chromosome 4 and segmental uniparental disomy of 9p24.3→p21.3 in GSCs. Gene expression profiling revealed 418 genes upregulated in tumor tissue vs. CD133pos./CD15pos. cells and 44 genes upregulated in CD133pos./CD15pos. cells vs. tumor tissue. Pathway analyses demonstrated that upregulated genes in CD133pos./CD15pos. cells are relevant to cell cycle processes and cancerogenesis. In summary, we detected previously undescribed genomic and gene expression differences when comparing tumor tissue and isolated stem-like subpopulations.

Combined Targeting of Glioblastoma Stem-Like Cells by Neutralizing RNA-Bio-Drugs for STAT3.

An important drawback in the management of glioblastoma (GBM) patients is the frequent relapse upon surgery and therapy. A likely explanation is that conventional therapies poorly affect a small population of stem-like cancer cells (glioblastoma stem cells, GSCs) that remain capable of repopulating the tumour mass. Indeed, the development of therapeutic strategies able to hit GSCs while reducing the tumour burden has become an important challenge to increase a patient’s survival. The signal transducer and activator of transcription-3 (STAT3) has been reported to play a pivotal role in maintaining the tumour initiating capacity of the GSC population. Therefore, in order to impair the renewal and propagation of the PDGFRβ-expressing GSC population, here we took advantage of the aptamer–siRNA chimera (AsiC), named Gint4.T-STAT3, that we previously have shown to efficiently antagonize STAT3 in subcutaneous PDGFRβ-positive GBM xenografts. We demonstrate that the aptamer conjugate is able to effectively and specifically prevent patient-derived GSC function and expansion. Moreover, because of the therapeutic potential of using miR-10b inhibitors and of the broad expression of the Axl receptor in GBM, we used the GL21.T anti-Axl aptamer as the targeting moiety for anti-miR-10b, showing that, in combination with the STAT3 AsiC, the aptamer–miR-10b antagonist treatment further enhances the inhibition of GSC sphere formation. Our results highlight the potential to use a combined approach with targeted RNA therapeutics to inhibit GBM tumour dissemination and relapse.

Deregulated expression of the imprinted DLK1-DIO3 region in glioblastoma stemlike cells: tumor suppressor role of lncRNA MEG3.

BackgroundGlioblastoma (GBM) stemlike cells (GSCs) are thought to be responsible for the maintenance and aggressiveness of GBM, the most common primary brain tumor in adults. This study aims at elucidating the involvement of deregulations within the imprinted delta-like homolog 1 gene‒type III iodothyronine deiodinase gene (DLK-DIO3) region on chromosome 14q32 in GBM pathogenesis.MethodsReal-time PCR analyses were performed on GSCs and GBM tissues. Methylation analyses, gene expression, and reverse-phase protein array profiles were used to investigate the tumor suppressor function of the maternally expressed 3 gene (MEG3).ResultsLoss of expression of genes and noncoding RNAs within the DLK1-DIO3 region was observed in GSCs and GBM tissues compared with normal brain. This downregulation is mainly mediated by epigenetic silencing. Kaplan–Meier analysis indicated that low expression of MEG3 and MEG8 long noncoding (lnc)RNAs significantly correlated with short survival in GBM patients. MEG3 restoration impairs tumorigenic abilities of GSCs in vitro by inhibiting cell growth, migration, and colony formation and decreases in vivo tumor growth, reducing infiltrative growth. These effects were associated with modulation of genes involved in cell adhesion and epithelial-to-mesenchymal transition (EMT).ConclusionIn GBM, MEG3 acts as a tumor suppressor mainly regulating cell adhesion, EMT, and cell proliferation, thus providing a potential candidate for novel GBM therapies.

Mir-370-3p Impairs Glioblastoma Stem-Like Cell Malignancy Regulating a Complex Interplay between HMGA2/HIF1A and the Oncogenic Long Non-Coding RNA (lncRNA) NEAT1

Glioblastoma (GBM) is the most aggressive and prevalent form of a human brain tumor in adults. Several data have demonstrated the implication of microRNAs (miRNAs) in tumorigenicity of GBM stem-like cells (GSCs). The regulatory functions of miRNAs in GSCs have emerged as potential therapeutic candidates for glioma treatment. The current study aimed at investigating the function of miR-370-3p in glioma progression, as aberrant expression of miR-370-3p, is involved in various human cancers, including glioma. Analyzing our collection of GBM samples and patient-derived GSC lines, we found the expression of miR-370-3p significantly downregulated compared to normal brain tissues and normal neural stem cells. Restoration of miR-370-3p expression in GSCs significantly decreased proliferation, migration, and clonogenic abilities of GSCs, in vitro, and tumor growth in vivo. Gene expression analysis performed on miR-370-3p transduced GSCs, identified several transcripts involved in Epithelial to Mesenchymal Transition (EMT), and Hypoxia signaling pathways. Among the genes downregulated by the restored expression of miR-370-3p, we found the EMT-inducer high-mobility group AT-hook 2 (HMGA2), the master transcriptional regulator of the adaptive response to hypoxia, Hypoxia-inducible factor (HIF)1A, and the long non-coding RNAs (lncRNAs) Nuclear Enriched Abundant Transcript (NEAT)1. NEAT1 acts as an oncogene in a series of human cancers including gliomas, where it is regulated by the Epidermal Growth Factor Receptor (EGFR) pathways, and contributes to tumor growth and invasion. Noteworthy, the expression levels of miR-370-3p and NEAT1 were inversely related in both GBM tumor specimens and GSCs, and a dual-luciferase reporter assay proved the direct binding between miR-370-3p and the lncRNAs NEAT1. Our results identify a critical role of miR-370-3p in the regulation of GBM development, indicating that miR-370-3p acts as a tumor-suppressor factor inhibiting glioma cell growth, migration and invasion by targeting the lncRNAs NEAT1, HMGA2, and HIF1A, thus, providing a potential candidate for GBM patient treatment.

Curcumin Loaded in Niosomal Nanoparticles Improved the Anti-tumor Effects of Free Curcumin on Glioblastoma Stem-like Cells: an In Vitro Study

Using a novel curcumin-loaded niosome nanoparticle (CM-NP), the present study was designed to evaluate the effect of curcumin on human glioblastoma stem-like cells (GSCs). CM-NP has a diameter of ~ 60 nm and a zeta potential of ~ − 35 mV with a constant physicochemical stability. The cytotoxic effects of free curcumin (CM) and CM-NP were investigated on GSCs obtained during the removal of a brain tumor. Both CM and CM-NP caused a dose-dependent decrease in cell proliferation and viability of GSCs. The IC50 values of CM and CM-NP on GSCs were 50 and 137 μg/ml after 24 h, respectively. CM-NP exerted significantly higher effects on GSC viability, apoptosis, cell cycle arrest, and the expression of Bax, a pro-apoptotic marker, compared with CM. In addition, the migration of GSCs was significantly impaired following the administration of CM-NP compared with CM. Furthermore, CM-NP significantly increased the values of reactive oxygen species and decreased the mRNA expressions of NF-κB and IL-6 of GSCs compared with CM. Our data also revealed that CM-NP could significantly reduce the invasiveness of GSCs compared with CM, possibly via MCP-1-mediated pathways. In addition, CM-NP exhibited a significantly greater inhibitory effect on colony formation of GSCs compared with CM. These data indicate that CM-NP exhibited stronger anti-tumor effects on GSCs than CM. Although further in vivo investigations are warranted, our results suggest that CM-NP could be an ideal carrier to deliver curcumin for potential therapeutic approaches into glioblastoma.