Neurosphere Cells Research Articles

AB029. Mitochondrial dysfunction and impaired growth of glioblastoma cell lines caused by antimicrobial agents inducing ferroptosis.

Glioblastoma, a malignant tumor, has no curative treatment. Mitochondria have been reported to be involved in tumorigenesis and drug resistance in various cancers. Recently, mitochondria have been considered a potential target for treating glioblastoma. We are developing a new treatment for glioblastoma targeting mitochondria. The microenvironment of glioblastoma is reported to be a low-nutrition and hypoxic condition. We focused our research on how mitochondria work under tumor circumstances. In this research, we used cell lines including U87, LN229, U373, T98G, and two patient-derived stem-like cells. First, we investigated the efficacy of mitochondria-targeted glioblastoma therapy in cell lines under glucose-starved conditions and the mechanism of cell death caused by mitochondria-targeted therapy. Second, we developed a treatment effective for cells with more glucose, because we believe that there is some lesion with higher glucose concentration in the tumor considering the heterogeneity of the tumor. We discovered that under glucose-starved conditions, mitochondria-related proteins and oxidative phosphorylation activity increased in glioblastoma cells, and in this condition, glioblastoma cells strongly depended on mitochondria. Administration of agents causing mitochondrial dysfunction including chloramphenicol was very effective and it led to cell death. The mechanism of cell death was not apoptosis but ferroptosis which is a recently discovered type of cell death caused by iron accumulation. In normoglycemic conditions, the effect of chloramphenicol was poor for the short term, however, for the long term, it was effective and caused cell death. Although both chloramphenicol alone and combined treatment using 2-DG (glycolysis inhibitor) and chloramphenicol had poor effects on cells in glucose-sufficient conditions, combined treatment was very effective for the short term under normal glucose conditions. These results suggest the importance of controlling glucose concentration. The effect of combined treatment was also confirmed in hypoxic conditions and for patient-derived neurosphere cells. And the mechanism of cell death was ferroptosis, too. Our mitochondria-targeted treatment was effective for cells with both glucose-starved conditions and normoglycemic conditions. It is necessary to investigate its effectiveness in vivo in the future. However, chloramphenicol has already been used, and therefore, this treatment can be used for humans safely. This discovery is expected to make a major contribution to the development of treatments for glioblastomas.

DIPG-64. UNRAVELING H3K27M INTERACTIONS IN DIFFUSE MIDLINE GLIOMA

Abstract BACKGROUND Diffuse midline glioma (DMG) harboring the histone H3 K27M mutation, also known as diffuse intrinsic pontine glioma when it occurs in the pons, is a uniformly fatal pediatric brain tumor. We sought to better understand protein-protein interactions of the mutant histone in DMG. METHODS To address this, we carried out endogenous co-immunoprecipitation of histone H3 K27M from patient-derived DMG neurosphere cell lines, followed by LC-MS/MS identification of binding partners, and validation of binding by Western Blot. Moving beyond identification, we investigated the functional consequences of these interactions. RESULTS We identified putative novel interactors of H3 K27M with notable binding partners including histone chaperones and RNA helicases of the DDX family. We then explored the disruption of downstream effects resulting from this interaction by pharmacologically inhibiting DDX3 using the small molecule inhibitor RK-33, and assessed impact on viability and radiation sensitivity. CONCLUSIONS Understanding the protein interactome of the mutant histone can inform alternative ways for targeting DMG, thus enriching the therapeutic toolbox against this aggressive tumor. Functional characterization of binding partners holds the potential to identify novel therapeutic targets that cooperate with H3 K27M. The use of inhibitors targeting these partners could specifically destabilize oncohistone-dependent homeostasis and survival pathways and increase sensitivity to radiation and other therapies.

DIPG-87. TARGETING GENE-REPRESSIVE EPIGENETIC REGULATION TO INDUCE INTERFERON SIGNALING IN DIFFUSE MIDLINE GLIOMA

Abstract BACKGROUND Diffuse intrinsic pontine glioma (DIPG) is a fatal pediatric brainstem tumor with no known effective treatment. DIPG is driven by the H3K27M mutant histone, which promotes epigenetic dysregulation, leading to loss of gene-repressive H3K27me3 marks, global DNA hypomethylation, and a distinct gene expression profile. DIPG cells retain histone methylation at histone H3 lysine 9 (H3K9me2/3), suggesting that the tumor may rely on these marks to repress unwanted expression. Our prior work has demonstrated that treatment with the DNA hypomethylating agent decitabine (DAC) elicits expression of cancer-specific antigens and immune activation, including activation of interferon signaling, but modulating gene-repressive H3K9 methylation in DIPG has not been extensively investigated. METHODS We treated four primary patient-derived DIPG neurosphere cell lines with BIX01294 and UNC0642, inhibitors of H3K9 methyltransferases G9a/GLP, and analyzed gene expression changes by RNA-sequencing and Western blot. We assessed the effect of combination treatments on these four cell lines with both DAC and either BIX01294 or UNC0642 and evaluated impact on gene expression, innate immune signaling, and interferon pathway activation. RESULTS We found that treatment with either of the G9a/GLP inhibitors resulted in the upregulation of genes involved in immune signaling. Given the similarities in the immune signaling response elicited from treatment with DAC and the G9a/GLP inhibitors, we evaluated the effect of combination treatments with both DAC and either BIX01294 or UNC0642. These combinations resulted in an increased induction of genes involved in immune signaling, including STING. CONCLUSIONS Pharmacologic inhibition of DNA methylation and histone H3K9 methylation elicits immune activation in DIPG cells. Upregulation of interferon and STING signaling with this combination treatment may be significant in enhancing the immunogenicity of DIPG cells to allow effective immunotherapy.

DIPG-58. TARGETING DISORDERED DNA METHYLATION IN DIPG TO CONSTRAIN VARIABILITY AND INDUCE IMMUNE SIGNALING

Abstract BACKGROUND Diffuse Intrinsic Pontine Glioma (DIPG) is a universally fatal pediatric brain cancer characterized by the histone H3 K27M mutation. The known downstream consequences of this mutation include loss of repressive chromatin marks, global DNA hypomethylation, and altered gene expression. METHODS We sought to investigate the role of epigenetic variability as a basis of DIPG cellular heterogeneity and plasticity by using Whole Genome Bisulfite Sequencing (WGBS). Existing methods of analysis of WGBS are limited in that they average/smooth methylation data and fail to capture intrinsic variability. We performed (WGBS) on 23 primary patient samples of DIPG and applied an analysis that captures the variability of DNA methylation reads to characterize methylation stochasticity. We then sought to modulate the epigenome pharmacologically using a DNA methyltransferase inhibitor and assessed changes through RNA-Seq and immunopeptidomics. RESULTS We find that DIPG has a marked increase in methylation stochasticity (quantified as methylation entropy). Additionally, while DIPG is globally hypomethylated, it retains DNA methylation at specific regulatory regions, such as bivalent promoters, and key genes, such as FOXG1, CXCR4, and KLF4. We sought to reverse these changes using the DNA methyltransferase inhibitor, decitabine, in 4 DIPG neurosphere cell lines. Treatment with decitabine reversed the entropy seen in DIPG and induced innate immune signaling and interferon signaling in DIPG cells. Using RNA-seq, we found that decitabine treatment dramatically alters gene expression, including activation of endogenous retroviral elements, activation of interferon signaling, de-repression of hypermethylated targets, and expression of putative neoantigens, such as PRAME and DAZL. Immunopeptidomics profiling of MHC Class I bound canonical and non-canonical peptides of DIPG cell lines after decitabine treatment also revealed decitabine-induced differences. CONCLUSIONS Taken together, our study finds that the methylome of DIPG is highly disordered but is also highly responsive to pharmacologic modulation by hypomethylating agents. Furthermore, hypomethylating drugs can increase the immunogenicity of DIPG, thus offering the potential for future combination with immunotherapy.

The Gap Junction Inhibitor Octanol Decreases Proliferation and Increases Glial Differentiation of Postnatal Neural Progenitor Cells.

Neural precursor cells (NPCs) that persist in the postnatal/adult subventricular zone (SVZ) express connexins that form hemichannels and gap junctions. Gap junctional communication plays a role in NPC proliferation and differentiation during development, but its relevance on postnatal age remains to be elucidated. In this work we aimed to evaluate the effect of the blockade of gap junctional communication on proliferation and cell fate of NPCs obtained from the SVZ of postnatal rats. NPCs were isolated and expanded in culture as neurospheres. Electron microscopy revealed the existence of gap junctions among neurosphere cells. Treatment of cultures with octanol, a broad-spectrum gap junction blocker, or with Gap27, a specific blocker for gap junctions formed by connexin43, produced a significant decrease in bromodeoxyuridine incorporation. Octanol treatment also exerted a dose-dependent antiproliferative effect on glioblastoma cells. To analyze possible actions on NPC fate, cells were seeded in the absence of mitogens. Treatment with octanol led to an increase in the percentage of astrocytes and oligodendrocyte precursors, whereas the percentage of neurons remained unchanged. Gap27 treatment, in contrast, did not modify the differentiation pattern of SVZ NPCs. Our results indicate that general blockade of gap junctions with octanol induces significant effects on the behavior of postnatal SVZ NPCs, by reducing proliferation and promoting glial differentiation.

DNA Methylation Profiles Are Stable in H3 K27M-Mutant Diffuse Midline Glioma Neurosphere Cell Lines.

Diffuse midline gliomas are among the deadliest human cancers and have had little progress in treatment in the last 50 years. Cell cultures of these tumors have been developed recently, but the degree to which such cultures retain the characteristics of the source tumors is unknown. DNA methylation profiling offers a powerful tool to look at genome-wide epigenetic changes that are biologically meaningful and can help assess the similarity of cultured tumor cells to their in vivo progenitors. Paraffinized diagnostic tissue from three diffuse intrinsic pontine gliomas with H3 K27M mutations was compared with subsequent passages of neurosphere cell cultures from those tumors. Each cell line was passaged 3-4 times and analyzed with DNA methylation arrays and standard algorithms that provided a comparison of diagnostic classification and cluster analysis. All samples tested maintained high classifier scores and clustered within the reference group of H3 K27M-mutant diffuse midline gliomas. There was a gain of 1q in all cell lines, with two cell lines initially manifesting the gain of 1q only during culture. In vitro cell cultures of H3 K27M-mutant gliomas maintain high degrees of similarity in DNA methylation profiles to their source tumor, confirming their fidelity even with some chromosomal changes.

Econazole selectively induces cell death in NF1-homozygous mutant tumor cells

Cutaneous neurofibromas (cNFs) are tumors that develop in more than 99% of individuals with neurofibromatosis type 1 (NF1). They develop in the dermis and can number in the thousands. cNFs can be itchy and painful and negatively impact self-esteem. There is no US Food and Drug Administration (FDA)-approved drug for their treatment. Here, we screen a library of FDA-approved drugs using a cNF cell model derived from human induced pluripotent stem cells (hiPSCs) generated from an NF1 patient. We engineer an NF1 mutation in the second allele to mimic loss of heterozygosity, differentiate the NF1+/- and NF1-/- hiPSCs into Schwann cell precursors (SCPs), and use them to screen a drug library to assess for inhibition of NF1-/- but not NF1+/- cell proliferation. We identify econazole nitrate as being effective against NF1-/- hiPSC-SCPs. Econazole cream selectively induces apoptosis in Nf1-/- murine nerve root neurosphere cells and human cNF xenografts. This study supports further testing of econazole for cNF treatment.

P07.02.B A FATTY-ACID BASED PROBE FOR NEAR INFRARED FLUORESCENCE GUIDED SURGERY OF GLIOBLASTOMA

Abstract BACKGROUND Glioblastoma (GBM) is the most aggressive primary tumor of the brain with a median survival of 15 months. Maximal safe resection is the initial standard-of-care, but resection is limited by the diffuse invasiveness of the tumor. Fluorescence imaging has been shown to aid intra-operative tumor identification, but the current options fail to do so consistently. Recent literature has shown fatty acid (FA) metabolism and uptake to be altered in glioblastoma. For this reason, we introduce a novel, near-infrared fluorescent dye, fatty-acid indocyanine green (FA-ICG), that aims to target the enhanced fatty acid uptake of glioblastoma. We evaluate this dye in in vitro and in vivo models. MATERIAL AND METHODS FA-ICG consists of palmitic acid, a long-chain, saturated fatty acid, that has been covalently linked to indocyanine green (ICG), as described previously. We validated FA-ICG in vitro in one animal cell line and four (two primary and two recurrent) patient-derived neurosphere glioblastoma cell lines. We performed in vitro temporal uptake dynamics and oleic acid based inhibition assays for FA-ICG and ICG on all cell lines. Time uptake was investigated by starving and then incubating cells with either dye for an increasing length of time. Similarly, inhibition was tested by incubating cells with increasing concentrations of oleic acid (in addition to either FA-ICG or ICG). Subsequently, the dye was tested in an NRMI mouse model without tumor for pharmacokinetic properties. Mice were injected with the dye and blood was collected at multiple time points post-injection. Quantification of fluorescence signals in serum was performed using a fluorescent standard curve. RESULTS FA-ICG was taken up by all glioblastoma cells lines. Across all cell cultures, uptake of FA-ICG showed a stepwise increase of up to 5-30 times higher uptake (compared to uptake at baseline) when cells were starved longer, while ICG uptake showed the same trend in only three out of five cell lines (and only up to 5 times higher uptake than at baseline). Oleic acid was found to inhibit FA-ICG uptake by 40% at low concentrations and up to 80-90% at higher concentrations across all cell lines, while ICG was unaffected. In vivo pharmacokinetics showed FA-ICG to have a mean half-life of 65 minutes in blood. CONCLUSION Our findings indicate that the uptake of FA-ICG is affected by starvation and oleic acid competition across all cell lines while ICG uptake is only partially affected by starvation. In vivo pharmacokinetic shows the half-life of FA-ICG to be significantly longer than that of ICG in literature. Taken together, these findings might point to an enhanced targeting and retention mechanism of FA-ICG in glioblastoma. Therefore, these results support the further translation of FA-ICG for fluorescence guided surgery in GBM.

DIPG-42. MAPPING THE DNA METHYLATION LANDSCAPE OF DIPG REVEALS NOTABLE METHYLOME VARIABILITY RESPONSIVE TO PHARMACOLOGIC MODULATION

Abstract Diffuse intrinsic pontine glioma (DIPG) is an incurable pediatric brainstem tumor, driven by the H3K27M mutation leading to epigenetic dysregulation. Epigenomic analyses of DIPG have shown global loss of repressive chromatin marks accompanied by DNA hypomethylation and altered gene expression. However, most investigations of the epigenetic landscape of DIPG only provide a static view of the epigenome. Such studies are not capable of adequately capturing the regulatory underpinnings of DIPG intratumoral heterogeneity and phenotypic plasticity. To address this, we performed Whole-Genome Bisulfite Sequencing (WGBS) on 23 primary patient DIPG specimens, 4 patient-derived DIPG neurosphere cell lines, and 5 normal controls. To analyze this data set, we applied a novel framework for analysis of DNA methylation variability, called informME analysis, which permits the derivation of comprehensive genome-wide DNA methylation potential energy landscapes that capture intrinsic epigenetic variation. We show that DIPG exhibits a markedly disordered epigenome, with increased stochasticity localizing to key regulatory elements and genes. We demonstrate that epigenetic instability maps onto key genes regulating pluripotency and developmental identity and hypothesize that these changes might enable tumor cells to sample a diverse range of transcriptional programs and phenotypes. Given this, we evaluated the responsiveness of the DIPG epigenetic landscape to pharmacologic modulation. We found that treatment with the DNA hypomethylating agent decitabine produced profound genome-wide demethylation and reduced DNA methylation stochasticity at active enhancers, bivalent promoters, and promoters of key developmental genes. Decitabine treatment also elicited changes in gene expression, including upregulation of genes related to immune signaling. Finally, treatment with DAC sensitized DIPG cells to the effects of histone deacetylase inhibition. This study suggests the application of epigenetic therapies to constrain cellular heterogeneity and plasticity in DIPG.

Differentiation of neurosphere after transplantation into the damaged spinal cord.

This study aimed to compare the differentiation and survival of human neural stem/progenitor cells of various origins in vitro and after transplantation into the injured spinal cord of laboratory animals. Rats with simulated spinal cord injury were transplanted with neurosphere cells obtained by directed differentiation of HUES6 cell lines. Fluorescence microscopy was used to visualize the obtained results. HUES6#1 and iPSC#1 neurospheres showed a wide range of markers associated with glial differentiation. The expression of the proliferation marker Ki67 did not exceed 25%, both in the lines of early and late neurospheres. Although neurospheres did not fully differentiate into astrocytes in vitro, they massively approached the GFAP+ astrocyte phenotype when exposed to the transplanted environment. PSC-derived neurospheres transplanted into the site of SM injury without additional growth factors showed only moderate survival, a significant degree of differentiation into astrocytes, and moderate differentiation into neurons. The difference in the survival and differentiation of HUES6#1 and iPSC#1 neurospheres, both in vitro and in vivo, can be explained by the difference in the regulatory behavior of signaling molecules corresponding to the source of origin of PSCs. Derivatives of human PSCs of various origins obtained according to the described differentiation protocol did not mature into astrocytic populations, nor did the glycogenic transition of PSC-derived NSCs occur in vitro. The study demonstrated the impact of the injured spinal cord microenvironment on the differentiation of transplanted HUES6#1 and iPSC#1 into astrocytes. The results showed that HUES6-derived neurospheres generated 90% of GFAP+ astrocytes and 5-10% of early neurons, while iPSC-derived neurospheres generated an average of 74% GFAP+ astrocytes and 5% of early neurons in vivo.

Abstract 2162: Preliminary analysis of circulating biomarkers of glioblastoma response during chemoradiotherapy

Abstract Intro. Glioblastoma is the most common and aggressive adult brain cancer, with a 5-year survival of less than 15%. Treatment decisions are guided by imaging, which at early timepoints cannot distinguish tumor pseudoprogression from non-response. Our work seeks to identify circulating biomarkers that can classify response as an alternative or adjunct to imaging. Here we present preliminary data exploring the potential of circulating cytokines and tumor cells collected before, during, and after treatment with the goal to identify potential makers for treatment adaptation. Methods. After consent, patient blood was collected the week before initiating chemoradiotherapy, weekly during treatment, and three weeks after treatment. Serum was tested by multiplex bead-based immunoassay (Legendplex Human Inflammation Panel 1, Biolegend inc.) targeting 13 cytokines and ELISA targeting soluble Human GFAP (Glial fibrillary acidic protein) (Biovendor R&D). The Circulogix filtration system was also used to capture cells of size consistent with circulating tumor cells (CTCs). Initial efforts focused on cells co-staining for GFAP and Olig2 and negative for CD45 (hematopoietic marker). All patient tumor specimens and cell lines were verified for GFAP and Olig2 expression by IHC. We selected 13 patients for the current analysis: 5 with growth due to non-response (NR), 6 stable disease (S) and two pseudo progression (PP). These classifications were evaluated radiologically at the first follow up after chemoradiotherapy (S or no) and based on whether changes observed stabilized or resolved spontaneously within six months or continued to progress (NR or PP). Results. The immunoassays detected approximately 70% of the analytes in our samples. Among those, IL10 and IL33 were higher before treatment on patients later determined to have stable disease (IL10 p=0.09; IL33 p=0.043 KW test; IL10 median S=22.93 ng/mL, NR=13.415ng/mL; IL33 median S= 164.10ng/mL, NR=55.57ng/mL). Interestingly, IL33 is reduced on stable patients during week 5 of treatment (PRE vs. W5 p=0.033; median PRE=164.1ng/mL, W5=57.57ng/mL). GFAP presented a slightly lower level after treatment in stable patients (S vs. NR p=0.014; median S=0.441 ng/ml, NR=0.473 ng/ml). As proof of concept, 50 GBM cells from each of U87 adherent cells, or 0913 and 0821 neurosphere cells (CD45 negative) glioblastoma cell lines were mixed in separate vials with 106 hematopoietic cells (RAJI; CD45 positive) and filtered per the procedure to identify CTCs in patient blood. We separated GBM cells from hematopoietic cells on the filtration system. Conclusion. Our preliminary data suggest that blood obtained during chemoradiotherapy can identify biomarkers associated with response. For example, IL10, IL33, GFAP, and CTCs might be used to classify treatment response at very early timepoints. We continue to recruit additional patients for additional analyses. Citation Format: Paulo Roberto Del Valle, Jonathan B. Bell, Scott M. Welford, Kaylie Kullison, Alessandro Valderrama, Gregory A. Azzam, Jessica J. Meshman, Macarena I. De La Fuente, Eric A. Mellon. Preliminary analysis of circulating biomarkers of glioblastoma response during chemoradiotherapy [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 2162.

The Efficiency of Neurospheres Derived from Human Wharton's Jelly Mesenchymal Stem Cells for Spinal Cord Injury Regeneration in Rats.

Spinal cord injury (SCI) causes inflammation and neuronal degeneration, resulting in functional movement loss. Since the availability of SCI treatments is still limited, stem cell therapy is an alternative clinical treatment for SCI and neurodegenerative disorders. Human umbilical cord Wharton's jelly-derived mesenchymal stem cells (hWJ-MSCs) are an excellent option for cell therapy. This study aimed to induce hWJ-MSCs into neural stem/progenitor cells in sphere formation (neurospheres) by using neurogenesis-enhancing small molecules (P7C3 and Isx9) and transplant to recover an SCI in a rat model. Inducted neurospheres were characterized by immunocytochemistry (ICC) and gene expression analysis. The best condition group was selected for transplantation. The results showed that the neurospheres induced by 10 µM Isx9 for 7 days produced neural stem/progenitor cell markers such as Nestin and β-tubulin 3 through the Wnt3A signaling pathway regulation markers (β-catenin and NeuroD1 gene expression). The neurospheres from the 7-day Isx9 group were selected to be transplanted into 9-day-old SCI rats. Eight weeks after transplantation, rats transplanted with the neurospheres could move normally, as shown by behavioral tests. MSCs and neurosphere cells were detected in the injured spinal cord tissue and produced neurotransmitter activity. Neurosphere-transplanted rats showed the lowest cavity size of the SCI tissue resulting from the injury recovery mechanism. In conclusion, hWJ-MSCs could differentiate into neurospheres using 10 µM Isx9 media through the Wnt3A signaling pathway. The locomotion and tissue recovery of the SCI rats with neurosphere transplantation were better than those without transplantation.

Culture Protocol and Transcriptomic Analysis of Murine SVZ NPCs and OPCs.

The mammalian adult brain contains two neural stem and precursor (NPC) niches: the subventricular zone [SVZ] lining the lateral ventricles and the subgranular zone [SGZ] in the hippocampus. From these, SVZ NPCs represent the largest NPC pool. While SGZ NPCs typically only produce neurons and astrocytes, SVZ NPCs produce neurons, astrocytes and oligodendrocytes throughout life. Of particular importance is the generation and replacement of oligodendrocytes, the only myelinating cells of the central nervous system (CNS). SVZ NPCs contribute to myelination by regenerating the parenchymal oligodendrocyte precursor cell (OPC) pool and by differentiating into oligodendrocytes in the developing and demyelinated brain. The neurosphere assay has been widely adopted by the scientific community to facilitate the study of NPCs in vitro. Here, we present a streamlined protocol for culturing postnatal and adult SVZ NPCs and OPCs from primary neurosphere cells. We characterize the purity and differentiation potential as well as provide RNA-sequencing profiles of postnatal SVZ NPCs, postnatal SVZ OPCs and adult SVZ NPCs. We show that primary neurospheres cells generated from postnatal and adult SVZ differentiate into neurons, astrocytes and oligodendrocytes concurrently and at comparable levels. SVZ OPCs are generated by subjecting primary neurosphere cells to OPC growth factors fibroblast growth factor (FGF) and platelet-derived growth factor-AA (PDGF-AA). We further show SVZ OPCs can differentiate into oligodendrocytes in the absence and presence of thyroid hormone T3. Transcriptomic analysis confirmed the identities of each cell population and revealed novel immune and signalling pathways expressed in an age and cell type specific manner.

Establishment of neural stem cell culture from the central nervous system of the Iberian ribbed newt Pleurodeles waltl.

Urodele amphibians have exceptional regeneration ability in various organs. Among these, the Iberian ribbed newt (Pleurodeles waltl) has emerged as a useful model organism for investigating the mechanisms underlying regeneration. Neural stem cells (NSCs) are an important source of regeneration in the central nervous system (CNS) and their culture method in vitro has been well established. NSCs form spherical cell aggregates called neurospheres and their formation has been demonstrated in various vertebrates, including some urodele species, but not in P. waltl. In this study, we reported neurosphere formation in brain- and spinal cord-derived cells of post-metamorphic P. waltl. These neurospheres showed proliferative activity and similar expression of marker proteins. However, the surface morphology was found to vary according to their origin, implying that the characteristics of the neurospheres generated from the brain and spinal cord could be similar but not identical. Subsequent in vitro differentiation analysis demonstrated that spinal cord-derived neurospheres gave rise to neurons and glial cells. We also found that cells in neurospheres from P. waltl differentiated to oligodendrocytes, whereas those from axolotls were reported not to differentiate to this cell type under standard culture conditions. Based on our findings, implantation of genetically modified neurospheres together with associated technical advantages in P. waltl could reveal pivotal gene(s) and/or signaling pathway(s) essential for the complete spinal cord regeneration ability in the future.

Transcriptome Changes in Glioma Cells Cultivated under Conditions of Neurosphere Formation.

Glioma is the most common and heterogeneous primary brain tumor. The development of a new relevant preclinical models is necessary. As research moves from cultures of adherent gliomas to a more relevant model, neurospheres, it is necessary to understand the changes that cells undergo at the transcriptome level. In the present work, we used three patient-derived gliomas and two immortalized glioblastomas, while their cultivation was carried out under adherent culture and neurosphere (NS) conditions. When comparing the transcriptomes of monolayer (ML) and NS cell cultures, we used Enrichr genes sets enrichment analysis to describe transcription factors (TFs) and the pathways involved in the formation of glioma NS. It was observed that NS formation is accompanied by the activation of five common gliomas of TFs, SOX2, UBTF, NFE2L2, TCF3 and STAT3. The sets of transcripts controlled by TFs MYC and MAX were suppressed in NS. Upregulated genes are involved in the processes of the epithelial–mesenchymal transition, cancer stemness, invasion and migration of glioma cells. However, MYC/MAX-dependent downregulated genes are involved in translation, focal adhesion and apical junction. Furthermore, we found three EGFR and FGFR signaling feedback regulators common to all analyzed gliomas—SPRY4, ERRFI1, and RAB31—which can be used for creating new therapeutic strategies of suppressing the invasion and progression of gliomas.

NAMPT Inhibition Induces Neuroblastoma Cell Death and Blocks Tumor Growth.

High-risk neuroblastoma (NB) portends very poor prognoses in children. Targeting tumor metabolism has emerged as a novel therapeutic strategy. High levels of nicotinamide-adenine-dinucleotide (NAD+) are required for rapid cell proliferation. Nicotinamide phosphoribosyl transferase (NAMPT) is the rate-limiting enzyme for NAD+ salvage and is overexpressed in several cancers. Here, we determine the potential of NAMPT as a therapeutic target for NB treatment. NAMPT inhibition cytotoxicity was determined by trypan blue exclusion and LDH assays. Neuroblastoma stem cell self-renewal was evaluated by neurosphere assay. Protein expression was evaluated via Western blot. The effect of targeting NAMPT in vivo was determined using an NB1691-xenografted mouse model. Robust NAMPT expression was demonstrated in multiple N-MYC amplified, high-risk neuroblastoma cell lines. NAMPT inhibition with STF-118804 (STF) decreased ATP, induced apoptosis, and reduced NB stem cell neurosphere formation. STF treatment down-regulated N-MYC levels and abrogated AKT activation. AKT and glycolytic pathway inhibitors in combination with NAMPT inhibition induced robust, greater-than-additive neuroblastoma cell death. Lastly, STF treatment blocked neuroblastoma tumor growth in mouse xenograft models. NAMPT is a valid therapeutic target as inhibition promoted neuroblastoma cell death in vitro and prevented tumor growth in vivo. Further investigation is warranted to establish this therapy’s role as an adjunctive modality.

Abstract 908: Small RNA-seq analysis of PIWI-interacting RNAs in glioblastoma stem cells: Identification of new therapeutic targets in glioblastoma patients

Abstract Introduction: Glioblastoma (GBM) is the most common malignancy affecting the CNS. Despite radical therapy, recurrence of GBM is a frequent and relatively early event in the course of the disease. One of the putative mechanisms of early recurrence of GBM is the existence of glioblastoma stem cells (GSCs) that can resist therapy and subsequently establish new GBM foci. PIWI-interacting RNAs (piRNAs), responsible for maintaining genome stability, may play a key role not only in the biology of germ and stem cells but also GSCs since their dysregulated expression has been described in several cancers, including GBM. Thus, the identification of specific piRNAs in GSCs could allow the distinction of these cells from other GBM cells and their eventual targeting. Some piRNAs could also be promising biomarkers useful for prognosis and prediction of disease progression. Material and Methods: Native GBM tissues dissociated using the Papain Dissociation System were used for culturing of paired cell primary cultures. Cells were divided into two aliquots with different culture conditions. GSCs were cultured in DMEM/F12 medium supplemented with bFGF and EGF growth factors. DMEM medium containing 10% fetal bovine serum (FBS) was used to culture non-stem GBM cells. Subsequently, the presence of neural stem cell markers CD133 and SOX2, the ability of GSCs to establish tumors in immunodeficient mice, and the ability to differentiate into more mature cell types were analyzed. RNA isolated from the paired primary cultures was used for small-RNA sequencing (RNA-seq). QIAseq miRNA Library Kit was used for the preparation of cDNA libraries. Sequencing analysis was performed using the NextSeq 500/550 High Output v2 Kit (75 cycles) and the NextSeq 500 sequencer. Results: Twelve paired primary cultures were prepared to global piRNA expression profiling. Small RNA-seq bioinformatics analysis identified between 3.45 and 5.51% of unique mapped piRNA reads in all samples; GSCs expressing higher levels of piRNA molecules in comparison with paired GBM cells. Subsequently, we identified a set of significantly differentially expressed piRNAs in GSCs. Expression of selected piRNAs was then artificially regulated in primary GSCs in vitro, and the effect of these molecules on neurosphere formation, viability, and cell genome stability was analyzed. Finally, we also observed an association of piR-23231 with overall survival of GBM patients. All results will be presented at the AACR Annual Meeting 2022. Conclusion: Based on the results so far, piRNAs seem to play an important role in GSCs as well as GBM biology and their targeting may be a promising tool for therapy of GBM patients. The study was supported by the programme project of the Ministry of Health of the Czech Republic with reg. no. NV19-03-00501 and NV19-03-00559. Citation Format: Jiri Sana, Frantisek Siegl, Marek Vecera, Pavel Fadrus, Jaroslav Juracek, Petra Pokorna, Karolina Trachtova, Tomas Kazda, Ondrej Slaby. Small RNA-seq analysis of PIWI-interacting RNAs in glioblastoma stem cells: Identification of new therapeutic targets in glioblastoma patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 908.

Abstract PR013: Oct4 and Sox2 induce cellular transition of glioma stem cells to an immune suppressive, regulatory T cell-like state

Abstract Solid malignancies, including GBM, contain small subsets of cells that display stem-like properties (i.e. glioma stem cells or GSCs) and act as key determinants of therapeutic resistance and tumor recurrence. Mechanisms of GSC immune escape are considered fundamental to clinical GBM growth and recurrence. GBM cells escape antitumor immunity by modifying the tumor microenvironment by secreting immune-suppressive factors and recruiting anti-inflammatory/pro-oncogenic cells (e.g. T-regulatory cells, M2-like macrophages). However, whether subsets of GSCs can mimic T-reg-like functions by expressing proteins associated with T-reg immune-suppression remains unknown. Single-cell RNA sequencing analysis of GBM neurospheres revealed a previously unrecognized Oct4/Sox2high/FOXP3− cell subpopulation with high expression of TGFb1, CD39, CD73, PD-L1, and Galectin-1, a gene expression fingerprint typically associated with T-reg cells and their immune suppressive functions within the tumor microenvironment. Bioinformatics analysis of public databases shows that the above-mentioned genes are enriched in the mesenchymal GBM subtype and highly correlated with CD44 and TGFb type II receptor (TGFBR2) expression in both clinical GBM and primary GSCs. Mechanistically, Oct4 and Sox2 were found to directly induce TGFBR2 transcription concurrent with chromatin architectural changes (i.e. heterochromatin-to-euchromatin transition) involving the TGFBR2 gene. Transgenic TGFBR2 expression increased neurosphere cell growth capacity, upregulated expression of T-reg-like effector genes and enriched for CD44+ GSCs. Functionally, neurospheres expressing transgenic Oct4/Sox2 or transgenic TGFBR2 possess increased immunosuppressive capacity, and pharmacological inhibition of TGFBR2 depleted CD44+ GSCs and reduced immunosuppression in T-cell co-cultures. Taken together, we show that reprogramming events initiated by Oct4 and Sox2 induce a CD44+/FOX3P− GSC cell subset with a T-reg-like immunosuppressive transcriptional profile via a TGFBR2-dependent mechanism. This research provides the first description of such neoplastic cells in any malignancy and has high potential translational impact since targeting these tumor cell subsets and their immunosuppressive mechanisms may be critical to the successful development of GBM immunotherapies. Citation Format: Amanda Johnson, Jack Korleski, John Laterra, Hernando Lopez-Bertoni. Oct4 and Sox2 induce cellular transition of glioma stem cells to an immune suppressive, regulatory T cell-like state [abstract]. In: Proceedings of the AACR Special Conference on the Evolutionary Dynamics in Carcinogenesis and Response to Therapy; 2022 Mar 14-17. Philadelphia (PA): AACR; Cancer Res 2022;82(10 Suppl):Abstract nr PR013.

Hippocampals neurogenesis is impaired in mice with a deletion in the coiled coil domain of Talpid3-implications for Joubert syndrome.

Mutations in Talpid3, a basal body protein essential for the assembly of primary cilia, have been reported to be causative for Joubert Syndrome (JS). Herein, we report prominent developmental defects in the hippocampus of a conditional knockout mouse lacking the conserved exons 11 and 12 of Talpid3. At early postnatal stages, the Talpid3 mutants exhibit a reduction in proliferation in the dentate gyrus and a disrupted glial scaffold. The occurrence of mis-localized progenitors in the granule cell layer suggests a role for the disrupted glial scaffold in cell migration resulting in defective subpial neurogenic zone-to-hilar transition. Neurospheres derived from the hippocampus of Talpid3fl/flUbcCre mouse, in which Talpid3 was conditionally deleted, lacked primary cilia and were smaller in size. In addition, neurosphere cells showed a disrupted actin cytoskeleton and defective migration. Our findings suggest a link between the hippocampal defects and the learning/memory deficits seen in JS patients.

STEM-03. Oct4/Sox2 DRIVE AN IMMUNOSUPPRESSIVE GSC PHENOTYPE BY INDUCING T-REG EFFECTOR GENES VIA TGFBR2 SIGNALING

Abstract Solid malignancies, including GBM, contain small subsets of cells that display stem-like properties (i.e. glioma stem cells or GSCs) and act as key determinants of therapeutic resistance and tumor recurrence. Mechanisms of GSC immune escape are considered fundamental to clinical GBM growth and recurrence. GBM cells escape antitumor immunity by modifying the tumor microenvironment by secreting immune-suppressive factors and recruiting anti-inflammatory/pro-oncogenic cells (e.g. T-regulatory cells, M2-like macrophages). However, whether subsets of GSCs can mimic T-reg-like functions by expressing proteins associated with T-reg immune-suppression remains unknown. Single-cell RNA sequencing analysis of GBM neurospheres revealed a previously unrecognized Oct4/Sox2high/FOXP3- cell subpopulation with high expression of TGFb1, CD39, CD73, PD-L1, and Galectin-1, a gene expression fingerprint typically associated with T-reg cells and their immune suppressive functions within the tumor microenvironment. Bioinformatics analysis of public databases shows that the above-mentioned genes are enriched in the mesenchymal GBM subtype and highly correlated with CD44 and TGFb type II receptor (TGFBR2) expression in both clinical GBM and primary GSCs. Mechanistically, Oct4 and Sox2 were found to directly induce TGFBR2 transcription concurrent with chromatin architectural changes (i.e. heterochromatin-to-euchromatin transition) involving the TGFBR2 gene. Transgenic TGFBR2 expression increased neurosphere cell growth capacity, upregulated expression of T-reg-like effector genes and enriched for CD44+ GSCs. Functionally, neurospheres expressing transgenic Oct4/Sox2 or transgenic TGFBR2 possess increased immunosuppressive capacity, and pharmacological inhibition of TGFBR2 depleted CD44+ GSCs and reduced immunosuppression in Jurkat cell co-cultures. Taken together, we show that reprogramming events initiated by Oct4 and Sox2 induce a CD44+/FOX3P- GSC cell subset with a T-reg-like immunosuppressive transcriptional profile via a TGFBR2-dependent mechanism. This research provides the first description of such neoplastic cells in any malignancy and has high potential translational impact since targeting these tumor cell subsets and their immunosuppressive mechanisms may be critical to the successful development of GBM immunotherapies.