Neural Stem Cell Medium Research Articles

Generation of patient-derived pediatric pilocytic astrocytoma in-vitro models using SV40 large T: evaluation of a modeling workflow

PurposeAlthough pediatric low-grade gliomas (pLGG) are the most common pediatric brain tumors, patient-derived cell lines reflecting pLGG biology in culture are scarce. This also applies to the most common pLGG subtype pilocytic astrocytoma (PA). Conventional cell culture approaches adapted from higher-grade tumors fail in PA due to oncogene-induced senescence (OIS) driving tumor cells into arrest. Here, we describe a PA modeling workflow using the Simian Virus large T antigen (SV40-TAg) to circumvent OIS.Methods18 pLGG tissue samples (17 (94%) histological and/or molecular diagnosis PA) were mechanically dissociated. Tumor cell positive-selection using A2B5 was perfomed in 8/18 (44%) cases. All primary cell suspensions were seeded in Neural Stem Cell Medium (NSM) and Astrocyte Basal Medium (ABM). Resulting short-term cultures were infected with SV40-TAg lentivirus. Detection of tumor specific alterations (BRAF-duplication and BRAF V600E-mutation) by digital droplet PCR (ddPCR) at defined time points allowed for determination of tumor cell fraction (TCF) and evaluation of the workflow. DNA-methylation profiling and gene-panel sequencing were used for molecular profiling of primary samples.ResultsPrimary cell suspensions had a mean TCF of 55% (+/− 23% (SD)). No sample in NSM (0/18) and ten samples in ABM (10/18) were successfully transduced. Three of these ten (30%) converted into long-term pLGG cell lines (TCF 100%), while TCF declined to 0% (outgrowth of microenvironmental cells) in 7/10 (70%) cultures. Young patient age was associated with successful model establishment.ConclusionA subset of primary PA cultures can be converted into long-term cell lines using SV40-TAg depending on sample intrinsic (patient age) and extrinsic workflow-related (e.g. type of medium, successful transduction) parameters. Careful monitoring of sample-intrinsic and extrinsic factors optimizes the process.

Construction of brain-like spheroids containing endothelial tubular networks by an adhesive culture system

Spheroids which are composed of several types of cells have been widely studied in the pharmaceutical field as their structure and functions are similar to human organs. Three-dimensional brain-like tissues are one of the most important tissues for the development of medicines to treat brain diseases and for in vitro brain models. In this study, spheroids mainly containing neurons, astrocytes, and endothelial cells were fabricated using a novel 3D culture plate, “MicoCell™” to construct a brain mimicking tissue. Due to the multicavity structures of MicoCell, ∼102 of attached spheroids were fabricated in a single plate. Spheroids in MicoCell were attached onto a mild cell adhesive surface, allowing for easy immunostaining and microscopic observation. Spheroid formation was improved by adding a Rho-Kinase inhibitor during cultivation. Endothelial cells formed vascular network structures in spheroids and some parts of the vascular structures attached onto the bottom of a culture plate. Co-culture of multiple cell types required optimization of the culture medium during spheroid formation. The mixture of neural stem cell medium and endothelial growth medium showed good spheroid formation and a vascular network. These results indicated that our culture plates and brain mimicking spheroids would be a suitable candidate for pharmaceutical applications such as drug screening and for in vitro brain models.

In-vitro differentiation of human embryonic stem cells into spinal cord neural stem cells.

In-vitro differentiation of human embryonic stem cells into spinal cord neural stem cells (NSCs) can help researchers better understand the cellular processes associated with spinal cord development and regeneration, and provide therapeutic strategies for spinal cord disorders. However, effective and consistent methods for the generation of human spinal cord NSCs are rare. Objective of the study is to establish methods for the in-vitro induction and long-term maintenance of human spinal cord NSCs. H9 cells were treated with neural induction medium for 10 days under single-cell seeding condition, followed by treatment with neural maintenance medium and replacement with NSC medium after five passages. The identity of the generated cells was determined by immunofluorescence, immunoblotting, and cleavage under targets and tagmentation (CUT&Tag) assays. After the neural induction, OCT4, an embryonic stem cell marker, was significantly reduced, whereas NESTIN and PAX6, two NSC markers, were clearly increased. After the neural maintenance, most of the H9-derived cells consistently expressed NESTIN and PAX6 together with SOX1 and HOXC9, two spinal cord markers. The Homer known motif enrichment results of the CUT&Tag assay confirmed the expression of HOXC9 in the H9-derived spinal cord NSCs, which can be maintained for more than 40 days under an in vitro culture system. This study sheds new light on effective induction and maintenance of human spinal cord NSCs.

Abstract 318: Multi-omics to edge into precision medicine for DIPG

Abstract Effective targeted cancer therapies are limited due to lack of our understanding of disease pathogenesis at the molecular level. Diffuse intrinsic pontine glioma (DIPG) is an incurable pediatric brain tumor with 80% of DIPGs harboring H3F3A (H3.3) mutation that lead to a substitution of methionine for lysine at position 27 (K27M). The tumors bearing H3.3K27M mutation arise throughout the midline structures with global depletion of H3.3K27 me3 (trimethylation). These histone mutations modify the epigenome and alter oncogenic transcription, causing oncogenic insults to progenitor cells in early neurodevelopment (PMID: 30453529). To determine the reprogramming pathways in the cell context of H3.3K27M tumors, we conducted liquid chromatography-mass spectrometry-based proteomic and phosphoproteomic analysis on seven patient-derived DIPG cell lines that can be stably passaged in serum-free neural stem cell media and displayed distinct morphologies, growth rates and chromosome abnormalities (PMID: 21368213). Three normal neuronal stem cell lines were included as non-tumor brain cells for comparison. Pathway analysis identified 29 pathways that are significantly altered in DIPG compared to normal brain cells at both the protein abundance and phosphosite level. Notably, AKT and MAPK associated PI3K signaling, VEGF signaling, mTOR signaling, and HIF1a signaling were differentially active in H3.3K27M tumors compared to healthy control cell lines. We saw significantly higher activity of multiple kinases involved in axon guidance and cytoskeletal remodeling in DIPG, such as PTK2B, DYRK2, TTBK2 and MARK2. This is the first time to report an increased abundance and kinase activity of Pyk2 protein (coded by PTK2B), a close homologue of FAK and its associated signaling in DIPG. Overexpression and autophosphorylation of Pyk2 are required to stimulate glioma cell migration (PMIDs: 15967096, 18648907). Pyk2 has also been proposed to act in concert with Src to link Gi- or Gq-coupled receptors with the mitogen-activated protein (MAP) kinase signaling pathway (PMID:12960403). Because of the shared signaling across kinase pathways, targeting activated Pyk2 in DIPG may complement inhibitors of other dysregulated signaling networks in DIPG such as MAPK2, VEGFR, PI3K and Src. Our data also found that IL13RA2 was upregulated in DIPG; others have shown that increased expression of IL13RA2 is associated with poor prognosis in GBM (PMIDs: 32913543, 30366424). We conclude that for H3 K27M DIPG tumors, campaigns to target Pyk2, MAPK2, VEGFR, PI3K, Src and IL13Ra2 using small molecules that traverse the blood brain barrier loom as promising opportunities for drug development. Citation Format: Nanyun Tang, Kristin Leskoske, Krystine Garcia-Mansfield, Ritin Sharma, Hannah Tolson, Patrick Pirrotte, Michael E. Berens. Multi-omics to edge into precision medicine for DIPG [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 318.

Enrichment and Transcriptional Characterization of Stem Cells Isolated from Human Glioblastoma Cell Lines.

Glioblastomas (GBM) are the most frequent and aggressive brain tumors due to their recurrence and resistance to current therapies. These characteristics are associated with the presence of glioma stem cells (GSCs), mainly identified by the detection of the membrane antigens CD133 and CD15. The main source of GSCs has been biopsies of tumors. However, alternatives are sought from cell lines because more homogeneous populations can be obtained with high yields. This chapter describes a method for the enrichment and characterization of GSCs from cell lines derived from human GBM by selective culture with serum-free neural stem cell medium and growth factors. The technique offers alternatives for the enrichment and characterization of GSCs, that could contribute to a better understanding of the biology of GBMs.

Effects of progesterone on the cell number of gliomaspheres derived from human glioblastoma cell lines

AimsThe malignancy of the Glioblastomas (GBM), the most frequent and aggressive brain tumors, have been associated with the presence of glioma stem cells (GSCs) which can form gliomaspheres (GS) in vitro. Progesterone (P) increases the proliferation, migration, and invasion of GBM cell lines through the interaction with its intracellular receptor (PR). However, it is unknown if the PR is expressed and the possible effects of P in the formation/differentiation of GS. Main methodsGS were grown from U251 and U87 cell lines by selective culture with serum-free neural stem cell medium. GSCs were identified by the detection of Sox2, Ki67, Nestin, CD133, and CD15 by immunofluorescence. Additionally, the relative expression of PROM1, NES, SOX2, OLIG2, EZH2, BMI1 and PR genes was evaluated by RT-qPCR. The GS were treated with P, and the number of cells was quantified. By RT-PCR the βIII-TUB and GFAP differentiation genes were evaluated. Key findingsGS were maintained until passage four. The expression of all GSCs markers was significantly higher in GS as compared with the basal culture of U251 and U87 cells. We demonstrated for the first time that PR is expressed in GS and this expression was higher as compared with the U251 and U87 cells in basal conditions. Also, we observed that P increased the number of cells derived primary gliomaspheres (GS1) from the U251 line, as well as the expression of the neuronal differentiation marker βIII-TUB. SignificanceThese results suggest the participation of P in the growth of GSCs.

Novel strategy for manufacturing autologous dendritic cell/allogeneic tumor lysate vaccines for glioblastoma

BackgroundGlioblastoma, the most common primary malignant brain tumor, is nearly universally fatal by 5 years. Dendritic cell vaccines are promising but often limited clinically by antigen choice, dendritic cell potency, and/or manufacturing yield. We optimized vaccine manufacture, generating potent mature autologous dendritic cells pulsed with allogeneic glioblastoma lysates.MethodsPlatelet lysate-based supplement was used to establish human glioblastoma cell lines. Phenotype and genotype were assessed. An improved culture technique to generate mature dendritic cells from glioblastoma patients’ monocytes was developed. The ability of T cells stimulated with autologous dendritic cells pulsed with allogeneic glioblastoma cell lysate to kill HLA-A2-matched glioblastoma cells was assessed.ResultsGlioblastoma cell lines established with platelet lysate supplement grew faster and expressed more stem-like markers than lines grown in neural stem cell media or in the presence of serum. They expressed a variety of glioma-associated antigens and had genomic abnormalities characteristic of glioblastoma stable up to 15 doublings. Unlike standard culture techniques, our optimized technique produced high levels of mature dendritic cells from glioblastoma patients’ monocytes. Autologous T cells stimulated with mature dendritic cells pulsed with allogeneic glioblastoma cell line lysate briskly killed HLA-A2-matched glioblastoma cells.ConclusionsOur glioblastoma culture method provides a renewable source for a broad spectrum glioblastoma neoantigens while our dendritic cell culture technique results in more mature dendritic cells in glioblastoma patients than standard techniques. This broadly applicable strategy could be easily integrated into patient care.

Derivation of neural stem cells from human teratomas.

Human teratoma is a germ cell tumor that contains normal tissues (e.g., hair, skin or cartilage) differentiated from embryonal germ layers. Because of the feature of this tumor, we hypothesized that human teratomas contain multipotent stem cells that can develop into various non-cancerous normal tissues. In this study, we cultured neurospheres originally derived from a human infantile teratoma tissue, and the sphere cells were found to possess the characteristics of neural stem cells. Tumor tissues were obtained from an infantile immature teratoma at the time of surgical resection. In the primary cell culture, colonies were formed in two weeks and were individually cultured in serum-free conditioned neural stem cell medium (NSC medium). Colonies changed into spheres and grew in smooth round forms, or attached to the bottom of the dishes and extended processes and filaments around. Sphere cells were dissociated into single cells, and new spheres (secondary spheres) were formed in NSC medium. Cell differentiation was induced by culturing cells in serum-containing medium (differentiation medium), as cells spread and attached to the bottom of dishes and changed form. The expression of Nestin, Sox2, CXCR4, and (stem cell markers), β3-tubulin (a neural marker) GFAP (a glial marker) CNPase, SOX10 (oligodendrocyte markers) and NF-L in cells was analyzed by immunofluorescence and a Q-PCR. Nestin, SOX2, CXCR4 were abundant in both primary and secondary spheres. Neural and glial markers (β3-tubulin and GFAP, respectively) were increased in cells cultured in differentiation medium while stem cell markers were diminished. The oligodendrocyte markers SOX10 and CNPase were also found in both spheres and differentiated cells. In conclusion, spheres with the characteristics of neural stem cells were obtained from the primary culture of a human infantile teratoma. These spheres are considered to have the potential to undergo a natural course of neural development in humans.

Isolation and Culture of Embryonic Mouse Neural Stem Cells.

Neural stem cells (NSCs) are multipotent and can give rise to the three major cell types of the central nervous system (CNS). In vitro culture and expansion of NSCs provide a suitable source of cells for neuroscientists to study the function of neurons and glial cells along with their interactions. There are several reported techniques for the isolation of neural stem cells from adult or embryo mammalian brains. During the microsurgical operation to isolate NSCs from different regions of the embryonic CNS, it is very important to reduce the damage to the brain cells to obtain the highest ratio of live and expandable stem cells. A possible technique for stress reduction during isolation of these cells from the mouse embryo brain is the reduction of surgical time. Here, we demonstrate a developed technique for rapid isolation of these cells from the E13 mouse embryo ganglionic eminence. Surgical procedures include harvesting E13 mouse embryos from the uterus, cutting the frontal fontanelle of the embryo with a bent needle tip, extracting the brain from the skull, microdissection of the isolated brain to harvest the ganglionic eminence, dissociation of the harvested tissue in NSC medium to gain a single cell suspension, and finally plating cells in suspension culture to generate neurospheres.

Exosome-Mediated Ultra-Effective Direct Conversion of Human Fibroblasts into Neural Progenitor-like Cells

Exosomes, naturally secreted nanoparticles, have been introduced as vehicles for horizontal transfer of genetic material. We induced autologous exosomes containing a cocktail of reprogramming factors ("reprosomes") to convert fibroblasts into neural progenitor cells (NPCs). The fibroblasts were treated with ultrasound and subsequently cultured in neural stem cell medium for 1 day to induce the release of reprosomes composed of reprogramming factors associated with chromatin remodeling and neural lineage-specific factors. After being treated with reprosomes, fibroblasts were converted into NPCs (rNPCs) with great efficiency via activation of chromatin remodeling, so quickly that only 5 days were required for the formation of 1500 spheroids showing an NPC-like phenotype. The rNPCs maintained self-renewal and proliferative properties for several weeks and successfully differentiated into neurons, astrocytes, and oligodendrocytes in vitro and in vivo. Reprosome-mediated cellular reprogramming is simple, safe, and efficient to produce autologous stem cells for clinical application.

The Effect of Saffron Aquatic Extract and Crocin on the Differentiation of Neural Stem Cells Into Oligodendrocyte Precursor Cells

Background: Oligodendrocytes are myelin-producing cells, which develop from oligodendrocyte precursor cells (OPCs) and may be damaged in multiple sclerosis (MS) and traumatic brain injuries. Promotion of neural stem cell (NSC) differentiation into oligodendrocytes in vivo is a viable strategy to replace the lost myelinating cells and restore motor functions in such debilitating diseases. Crocus sativus L. or saffron has been used as a spice since ancient times. Saffron and its major active component, crocin, are well-known for their medicinal and neuroprotective activities. The current study aimed at assessing the effect of saffron extract and crocin on the differentiation of NSCs into OPCs. Methods: To isolate NSCs, 14-day embryonic rats cortices were cultured, using the neurosphere assay. NSCs were cultured in a complete NSC medium under 4 different treatment conditions: 1, Negative control group, 10 ng/mL of basic fibroblast growth factor (bFGF); 2, Positive control group, 10 ng/mL of bFGF plus 30 ng/mL of platelet-derived growth factor-AA (PDGF-AA); 3, Crocin group, crocin extract plus 10 ng/mL of bFGF; and 4, Saffron group, saffron extract plus 10 ng/mL of bFGF in 2 or 5 days. The level of Olig2 protein, an early OPC marker, was evaluated by flow cytometry 2 days after the treatment. The level of sox10 mRNA expression, involving in OPC maturation processes, was assessed in the study groups via quantitative real-time polymerase chain reaction (PCR), 5 days after the treatment. All the experiments were done in triplicate. The data were analyzed using one-way ANOVA with Graph Pad Prism version 6.01; P < 0.05 was considered as the level of significance. Results: The present data demonstrated that, similar to the positive control group, the rate of olig2+ cells in the crocin and saffron extract groups did not exceed 80% indicating that these components could significantly increase olig2 expression in NSC differentiation compared with that of the negative controls (P < 0.05). Moreover, the mean expression level of sox10 gene in the positive control, saffron and crocin groups was 1 ± 0.012, 0.72 ± 0.13, and 0.835 ± 0.015, respectively, which significantly higher than that of the negative control group (P < 0.05). Conclusions: It can be concluded that crocin and saffron are the proper alternatives for PDGF-AA in the enhancement of NSC differentiation into oligodendrocytes.

Abstract 2453: Neuroblastoma is bi-phasic and includes classical neuro-epithelial cells and chemo-resistant mesenchymal cells

Abstract Introduction Most high stage neuroblastoma initially respond to chemotherapy, but ultimately relapse as therapy-resistant tumor. The mechanisms driving relapse and resistance remain elusive. We investigated whether neuroblastoma tumors include divergent cell types that may underlie this plasticity. Experimental procedures Fresh tumor cells cultured in neural stem cell medium were analyzed by FACS, whole genome sequencing, Chip-seq, mRNA profiling, and motility and chemo-sensitivity assays. Inducible transgenes were used to test state-transitions. Tumors were analyzed by immunohistochemistry. Results New neuroblastoma cell lines always included two cell types, which share the same genetic defects but have highly divergent phenotypes. One type has a neuro-epithelial (NE) phenotype and expresses all classical neuroblastoma markers. The other type has a mesenchymal (MES) character, is motile and lacks all neuroblastoma markers. Immunohistochemistry (IHC) detected a small fraction of MES cells in most primary neuroblastoma. In four isogenic cell line pairs, we found that MES cells were more chemo-resistant than their NE-type counterparts. Indeed, comparison of primary neuroblastoma lesions before and after chemotherapy showed an accumulation of viable MES-type cells in post treatment samples. Moreover, comparison of primary, pre-treatment tumors with relapses emerging 4-5 years later in the same patients showed a strong enrichment for MES cells in the latter. As these data suggest a role for MES-type cells in relapse development, we analyzed their key regulatory pathways. The isogenic MES-NE cell line pairs showed consistent mRNA expression differences between both phenotypes, activating major signaling routes and transcription factors. Chip-seq identified divergent histone modifications. MES cells had high NOTCH pathway activity and PRRX1 expression. Induced expression of NOTCH or PRRX1 converted multiple NE-type cell lines into MES-type cells, including chemo-resistance. Further analysis of these routes reconstructed molecular wiring of MES-type cells. This identified key-players like MEK and PDGFRβ, which were successfully targeted by small molecules to specifically kill MES cells in vitro. Conclusions Our data suggest that neuroblastoma is a bi-phasic tumor. MES and NE cells differ in many characteristics, but can transdifferentiate into each other. MES and NE cells may correspond to developmental stages, i.e. mesenchymal migratory cells delaminated from the neural crest and more differentiated cells of the adrenergic lineage. MES cells accumulate after chemo-therapy and in relapses. They may survive classical therapy and over time seed relapses, that ultimately become heterogeneous again. Elimination of MES cells with small molecule inhibitors shows how cells with a potential key role in relapse development are amenable to therapy. Citation Format: Rogier Versteeg, Tim van Groningen, Linda J. Valentijn, Bart A. Westerman, Jan J. Molenaar, Ellen M. Westerhout, Mohamed Hamdi, Godelieve A. Tytgat, Jan Koster, Johan van Nes. Neuroblastoma is bi-phasic and includes classical neuro-epithelial cells and chemo-resistant mesenchymal cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2453.

Abstract PR08: Neuroblastoma is biphasic and includes classical neuroepithelial cells and chemoresistant mesenchymal cells

Abstract Introduction: Most high stage neuroblastoma initially respond to chemotherapy, but ultimately relapse as therapy-resistant tumor. The mechanisms driving relapse and resistance remain elusive. We investigated whether neuroblastoma tumors include phenotypically and functionally divergent subsets of tumor cells that may underlie its clinical plasticity. Experimental Procedures: Fresh tumor cells were cultured in neural stem cell medium and analyzed by FACS, whole genome sequencing, mRNA profiling, motility assays and chemo-sensitivity assays. Lentivirally transduced inducible gene constructs were used to test state-transitions. Immunohistochemistry was used to define cellular subtypes in tumors. Results: We observed that new neuroblastoma cell lines always include two phenotypically divergent cell types. Both types share the same genetic defects, but have highly divergent phenotypes. One cell type has a neuro-epithelial (NE) phenotype and expresses all classical and diagnostically used neuroblastoma markers. The other type has a mesenchymal (MES) character, lacks all neuroblastoma markers and is highly motile. At low frequency, both cell types can spontaneously transdifferentiate in vitro. Immunohistochemistry (IHC) of primary neuroblastoma detected a small fraction of MES cells in most tumors. To analyze the clinical relevance of MES-type cells, we investigated their sensitivity to chemotherapeutics used in neuroblastoma treatment. In four isogenic pairs, MES cells were more resistant to the drugs than their NE-type counterparts. We investigated whether the chemo-resistance of MES cells may operate in vivo. We analyzed a series of primary neuroblastoma tumors surgically removed immediately after chemotherapy. The viable cells in the post-therapy samples were strongly enriched in MES-type cells as compared to the pre-treatment tumors of the same patients. We also compared primary, pre-treatment tumors with relapses emerging 4-5 years later in the same patients. Most strikingly, also the relapsed neuroblastoma tumors were highly enriched for MES-type cells. As these data suggest a role for MES-type cells in development of therapy-resistant relapses, we analyzed their key regulatory pathways. mRNA profiling of isogenic MES-NE cell line pairs identified consistent mRNA expression differences between both phenotypes. Major signaling routes and transcription factors were highly differentially expressed. MES-type cells had high expression and activation of NOTCH pathway genes and expression of the homeobox gene PRRX1. Induced expression of NOTCH or PRRX1 transgenes in multiple NE-type cell lines converted them into MES-type cell lines, including chemo-resistance. Analysis of the changes in gene expression and activity downstream of NOTCH or PRRX1 allowed reconstruction of the molecular wiring of MES-type cells. This identified several drugable key-players, like MEK and PDGFRβ. Targeting of them with small-molecule inhibitors specifically killed MES cells in vitro. Conclusions: Our data suggest that neuroblastoma is a bi-phasic tumor. MES and NE cells have very different characteristics, but can transdifferentiate into each other. It is tempting to speculate that the MES- and NE-phenotypes recapitulate two developmental stages of neuroblasts: MES cells may correspond to the migrating cell type that has delaminated from the neural crest, while NE cells could correspond to the more differentiated cell in the target organs expressing markers of the adrenalin synthesis route. MES cells strongly accumulate after chemo-therapy and in relapses. They may survive classical therapy and over time seed relapses, that ultimately become heterogeneous again. Targeted elimination of MES cells with small molecule inhibitors shows how cells with a potential key role in relapse development are amenable to therapy. This abstract is also presented as Poster B30. Citation Format: Rogier Versteeg, Tim van Groningen, Bart A. Westerman, Jan J. Molenaar, Ellen M. Westerhout, Mohamed Hamdi, Godelieve A. Tytgat, Jan Koster, Johan van Nes. Neuroblastoma is biphasic and includes classical neuroepithelial cells and chemoresistant mesenchymal cells. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Pediatric Cancer Research: From Mechanisms and Models to Treatment and Survivorship; 2015 Nov 9-12; Fort Lauderdale, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(5 Suppl):Abstract nr PR08.

MTR-10PHARMACOLOGICAL NOTCH BLOCKADE IN GLIOMAS INDUCES AUTOPHAGY AND COMBINATION TREATMENT WITH AN AUTOPHAGY INHIBITOR INCREASES TUMOR CELL DEATH

BACKGROUND: Induction of autophagy has been identified as a potential mechanism for chemotherapeutic resistance, and its manipulation has increasingly been explored as a treatment strategy in many cancers. However, there is minimal data on the effects of the Notch pathway on autophagy in cancer, and none in gliomas. We examined if Notch pathway blockade using a gamma-secretase inhibitor (GSI) MRK003 would induce autophagy in glioma neurosphere lines and studied the effects of chloroquine (CQ), a late-stage autophagy inhibitor, in combination with the GSI. METHODS: 500,000 cells from 2 neurosphere lines (JHH520 and HSR-GBM1), maintained in Neural Stem Cell medium with EGF and FGF, were treated with DMSO, 0.5-5 µM levels of MRK003, and/or 10 µM CQ for 24-72 hours. Western blotting was performed to detect alterations in protein levels of LC3B, pAkt, Akt, p62 and cleaved PARP. MTS, Ki-67, Annexin V, cell cycle and soft agar assays were performed to look at the combinatorial effects of MRK003 and CQ. RESULTS: An LC3B-II band, suggesting induction of autophagy, was observed after Notch pathway blockade in a dose dependent manner in the 2 neurosphere lines. Reduction of pAkt was seen in GBM1, suggesting mTORC2 inhibition. Decreased growth (MTS assay both p <0.001), proliferation (Ki-67 assay: both p <0.05), increased apoptosis (increased sub-G1 population: JHH520 p <0.005, HSR-GBM1 p <0.05; increased Annexin V percentage: JHH520 p <0.05, HSR-GBM1 p <0.01, increased cleaved PARP protein expression), and decreased clonogenicity (soft agar assay) were seen after combination MRK003 and CQ treatment compared to DMSO. CONCLUSIONS: Induction of autophagy, possibly through mTORC2 inhibition, occurs after Notch inhibition in glioma neurospheres using a GSI. Inhibition of autophagy using CQ caused increased cytotoxicity. These results suggest a possible resistance mechanism to Notch inhibition in malignant gliomas and pave the way for a new treatment strategy.

Conversion of Adipose Tissue-Derived Mesenchymal Stem Cells to Neural Stem Cell-Like Cells by a Single Transcription Factor, Sox2.

Adipose tissue is an attractive source of easily accessible adult candidate cells for regenerative medicine. Adipose tissue-derived mesenchymal stem cells (ADSCs) have multipotency and strong proliferation and differentiation capabilities in vitro. However, as mesodermal multipotent stem cells, whether the ADSCs can convert into induced neural stem cells (NSCs) has so far not been demonstrated. In this study, we found that normally the naïve ADSCs cultured as either monolayer or spheres in NSC medium did not express Sox2 and Pax6 genes and proteins, and could not differentiate to neuron-like cells. However, when we introduced the Sox2 gene into ADSCs by retrovirus, they exhibited a typical NSC-like morphology, and could be passaged continuously, and expressed NSC specific markers Sox2 and Pax6. In addition, the ADSC-derived NSC-like cells displayed the ability to differentiate into neuron-like cells when switched to the differentiation culture medium, expressing neuronal markers, including Tuj1 and MAP2 genes and proteins. Our results suggest the ADSCs can be converted into induced NSC-like cells with a single transcription factor Sox2. This finding could provide another alternative cell source for cell therapy of neurological disorders.

The role of microRNA-30c in the self-renewal and differentiation of C6 glioma cells

BackgroundSphere formation, one method for identifying self-renewal ability, has been used to report that cancer stem-like cells exist in rat C6 glioma cells. Recent studies suggested that cancer stem-like cells share the stem cell properties of self-renewal and multipotent ability of neural stem cells and might be regulated by microRNAs (miRNAs). However, the mechanism of miRNA involvement in the sphere formation and neural differentiation abilities of cancer stem-like cells is poorly understood. ResultsWe found that miRNA-30c could assist in sphere formation of C6 cells under defined conditions in neural stem cell medium DMEM/F12-bFGF-EGF-B27. Moreover, overexpression of miRNA-30c might reduce 3-isobutyl-1-methylxanthine (IBMX)-induced neural differentiation, as the expression of neural markers, especially glial fibrillary acidic protein (GFAP), decreased. Further experiments revealed that miRNA-30c inhibited the IBMX-induced astrocyte differentiation via targeting the upstream genes and inactivating phosphorylation of STAT3 of the JAK-STAT3 pathway. Subsequently, the expression of GFAP was reduced and the number of astrocyte differentiation from C6 cells decreased. ConclusionsOur findings suggest that miRNA-30c could play a regulatory role in self-renewal and neural differentiation in C6 glioma cells.

Akt‑mediated phosphorylation of Oct4 is associated with the proliferation of stem‑like cancer cells.

Oct4 protein encoded by POU5F1 plays a pivotal role in maintaining the self-renewal of pluripotent stem cells; however, its presence in cancer cells remains controversial. In the present study, we provided evidence that the transcripts of authentic OCT4 gene (OCT4A) and its multiple pseudogenes were detected in a variety of cancer cell lines. A few major bands were also detected by western blotting using an anti-Oct4A monoclonal antibody. Moreover, an anti-Oct4-pT235 antibody was used to identify a band in the majority of the tested cancer cell lines that coincided with one of the anti-Oct4A bands which was decreasable by a specific shRNA. The Oct4-pT235 signals were also detected in human glioblastoma and liver cancer specimens by immunofluorescence microscopy and immunohistochemistry. U87 glioblastoma cells were cultured in a neural stem cell medium to induce the formation of neurospheres rich in stem-like cancer cells. The levels of Oct4-pT235 in the sphere cells were markedly increased compared to their monolayer parental cells, a result that was accompanied by upregulation of the PI3K-Akt pathway. Akti-1/2, a specific inhibitor of Akt, effectively reduced the level of Oct4-pT235 and attenuated the proliferation of U87 sphere cells. ITE, an agonist of the aryl hydrocarbon receptor, also significantly attenuated the Akt-mediated phosphorylation of Oct4 in glioblastoma and liver cancer cells, and reduced their tumorigenic potential in a xenograft tumor model. Taken together, we concluded that the Akt-mediated phosphorylation of Oct4A or its homolog protein was associated with the proliferation of stem-like cancer cells that may serve as a novel biomarker and drug target for certain types of cancer.

Activated T cells induce oligodendrocyte progenitor cells proliferation by releasing VEGF-A

Neuroinflammatory diseases are characterized by infiltration of lymphocytes into the CNS, followed by demyelination and axonal degeneration. Evidence suggests that inflammatory factors released from activated T-cells induce neurotoxicity and impair function of neural stem cells. However, the effect of activated T-cells on the function of oligodendrocyte progenitor cells (OPCs) is uncertain, partly due to the difficulty in obtaining adult human oligodendrocytes. In this study, we used iNSCs to obtain OPCs and determined the effect of inflammatory factors on OPCs. Neural stem cells were induced from adult peripheral CD34 + cells by transduction using Sendai virus encoding transcription factor constructs of sox2, oct3/4, c-myc and klf4 and incubation in neural stem cell medium. The resulting cells were differentiated into OPCs and then exposed to supernatants from activated T-cells. Cell proliferation was determined by EdU incorporation assay and CellQuanti-Blue assay. Cell cycle progression was studied by transfecting OPCs with Fluorescence Ubiquitination Cell Cycle Indicator (FUCCI). VEGF-A and its receptors were studied by RT-PCR and western-blot assay. We found that supernatants from activated T cells increased OPC proliferation by regulating cell cycle progression. While VEGF-A expression was increased in T cells after activation, immunodepletion of VEGF-A from activated T cell supernatants significantly attenuated activated T cells induced OPC proliferation. Furthermore, we found that VEGF receptor 2 (VEGF-R2) was expressed on OPC and specific inhibition of VEGF-R2 with siRNA or inhibitor also attenuated activated T cell supernatants induced OPC proliferation. The effects of T cells and VEGF-A on OPC proliferation were also confirmed in primary human fetal OPC cultures. These results indicate that OPCs generated from CD34-derived neural stem cells behave similarly as primary OPCs in response to treatment by activated T cells and could be used to study the physiological functions and pathogenesis of OPCs, providing a specific advantage especially by using patient specific blood samples. Further, using this model we found that VEGF-A released from activated T cells could enhance OPC proliferation through activating VEGF-R2, indicating a complicated role for VEGF-A in T-cell mediated neuroinflammatory disorders.

TLX activates MMP-2, promotes self-renewal of tumor spheres in neuroblastoma and correlates with poor patient survival.

Nuclear orphan receptor TLX (Drosophila tailless homolog) is essential for the maintenance of neural stem/progenitor cell self-renewal, but its role in neuroblastoma (NB) is not well understood. Here, we show that TLX is essential for the formation of tumor spheres in three different NB cell lines, when grown in neural stem cell media. We demonstrate that the knock down of TLX in IMR-32 cells diminishes its tumor sphere-forming capacity. In tumor spheres, TLX is coexpressed with the neural progenitor markers Nestin, CD133 and Oct-4. In addition, TLX is coexpressed with the migratory neural progenitor markers CD15 and matrix metalloproteinase-2 (MMP-2) in xenografts of primary NB cells from patients. Subsequently, we show the effect of TLX on the proliferative, invasive and migratory properties of IMR-32 cells. We attribute this to the recruitment of TLX to both MMP-2 and Oct-4 gene promoters, which resulted in the respective gene activation. In support of our findings, we found that TLX expression was high in NB patient tissues when compared with normal peripheral nervous system tissues. Further, the Kaplan–Meier estimator indicated a negative correlation between TLX expression and survival in 88 NB patients. Therefore, our results point at TLX being a crucial player in progression of NB, by promoting self-renewal of NB tumor-initiating cells and altering their migratory and invasive properties.

Abstract 995: Loss of ATRX decreases survival and improves response to DNA damaging agents in a novel mouse model of glioblastoma

Abstract Pediatric glioblastoma (GBM) remains one of the most difficult childhood tumors to treat, and most patients will die within the first two years of receiving this diagnosis. ATRX is a histone chaperone protein that is mutated primarily in adolescent GBMs. No previous animal model has demonstrated the effect of ATRX loss on GBM formation. In this study, we determined the contribution of ATRX knockdown to GBM formation and treatment response in a novel mouse model of GBM. Using the second-generation shRNA-mir library, we cloned an ATRX knockdown sequence into a plasmid with flanking sequences recognized by the Sleeping Beauty (SB) transposase for insertion into host genomic DNA. Glioblastomas were induced in mice using the SB transposase system injecting plasmids encoding luciferase, shp53 and NRAS, with or without shATRX, into the ventricle of neonatal mice. Uptake of plasmid DNA as well as development of intracranial tumors was monitored by bioluminescence. When animals showed symptoms of tumor burden they were euthanized and brains were processed for histological evaluation or placed in culture with neural stem cell media (with EGF and FGF supplementation). Tumors in both groups (with or without shATRX) showed histological hallmarks of human grade IV glioblastoma. The loss of ATRX was confirmed by IHC, and was specifically localized within tumors generated with the shATRX plasmid and not in the tumors generated with shp53 and NRAS alone, nor in the adjacent normal cortex. Notably, loss of ATRX reduced median survival of mice by 43% (p=0.012). Tissue was analyzed by FISH telomere probe as ATRX loss in human tumors is associated with alternative lengthening of telomeres (ALT). ATRX-deficient tumors were significantly more likely to show chromosomal aneuploidy (p=0.015) by telomere FISH. Cell lines generated from ATRX-deficient tumors were confirmed to have reduction of ATRX expression. Tumor cell lines (with and without ATRX loss) were plated, treated at 24 hours with intervention or control, and analyzed for viability at 72 hours. ATRX-deficient tumor cells were significantly (p≤0.005) more sensitive to DNA damaging agents, including: (1) 5-FU, (2) doxorubicin, (3) UV irradiation, and (4) adenoviral thymidine kinase with ganciclovir; with the notable exception of temozolomide (p=0.86), which is the standard of care for treatment of pediatric GBM. Loss of ATRX in a mouse model hastens glioblastoma formation and decreases survival. In addition, loss of ATRX leads to aneuploidy and improved response to DNA damaging agents, providing possible targeted therapies for tumors with this mutation. This mouse model prospectively validates ATRX as a tumor suppressor in pediatric GBM for the first time in an animal model; and provides a platform for analysis of relevant pathways and development of potential novel therapies. Supported by NIH/NINDS grants to MGC and PRL. Citation Format: Carl Koschmann, Alexandra Calinescu, Marta Dzaman, Rosie Lemons, Daniel Thomas, Maria G. Castro, Pedro R. Lowenstein. Loss of ATRX decreases survival and improves response to DNA damaging agents in a novel mouse model of glioblastoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 995. doi:10.1158/1538-7445.AM2014-995