bluuebunny/crossref_metadata_2025_split huggingface dataset:
STEM-14. NEAR-INSTANT DETECTION OF GLIOMA STEM CELLS IN LIVE HUMAN GBM-TISSUE
Authors: Persson Oscar , Neofytou Christina , Keane Lily , Jensdottir Margret , Joseph Bertrand , Hermanson Ola , Ilkhanizadeh Shirin 
Published: November 2022

Abstract
Emerging research suggests that failure to target glioma stem cells (GSCs) rather than the inability to remove tumors through surgery, radiation, or chemotherapy, explains the poor survival of GBM patients. In this study, a luminescent conjugated oligothiophene (LCO), named GlioStem (p-HTMI), is used for non-invasive and non-amplified real-time detection of GSCs in live human GBM-tissue. More than 90 patient samples were stained, quantified, and analyzed by fluorescent microscopy for the presence of Gliostem-positive (GS+) cells. Approximately 30 of the samples were FACS-sorted for GS+ and GS- cells where quantification by FACS could verify microscopy results. In addition, GS+ cells were shown to express significantly higher levels of stem cell markers (CD271, CD133, PDGFRa, CD44) in FACS-experiments. Bulk RNA sequencing of 7 GBM patient samples with paired GS+ and GS- sorted cells revealed that GS+ patient samples clustered together, whereas the GS- populations did not cluster together neither with each other nor with the GS+ populations. These data suggest a distinct heterogeneity in the GS- samples and a certain level of homogeneity regarding the GS+ populations, independent of intra-patient or patient-to-patient heterogeneity. Moreover, the GS+ samples were found to express significantly higher levels of stem cell markers including SOX10, OLIG1/2, and ASCL1, compared to the GS- samples. More specifically, the GS+ samples exhibited significantly higher expression of 35 genes associated with stemness compared to the GS- samples, with most markers being associated with pre-oligodendrocyte precursor cells (pre-OPCs) and pro-neural subtypes. Our results suggest that GlioStem is a versatile tool for near-instant and selective detection of GSCs in live tumor tissue. Detecting and eliminating these cells during tumor resection may therefore be an important aim in efficiently preventing tumor regrowth and would mean a crucial step towards increased patient survival.
STEM-19. LIVE DETECTION OF NEURAL STEM AND GLIOBLASTOMA CELLS BY A LUMINESCENT CONJUGATED OLIGOTHIOPHENE DERIVATIVE
Authors: Ilkhanizadeh Shirin , Gracias Aileen , Åslund Andreas K O , Bäck Marcus , Simon Rozalyn , Neofytou Christina , Rraklli Vilma , Migliori Bianca , Kavanagh Edel , Nelander Sven , Westermark Bengt , Uhrbom Lene , Forsberg-Nilsson Karin , Teixeira Ana I , Konradsson Peter , Uhlén Per , Holmberg Johan , Joseph Bertrand , Nilsson K Peter R , Hermanson Ola 
Published: November 2022

Abstract
Glioblastoma (GBM) is an aggressive nervous system tumor with a mean survival time of 12-14 months. Cells with neural stem cell-like properties can be derived from GBM tumors. These cells seem to escape conventional irradiation treatment, chemotherapy, and surgery, and may play a crucial role for relapse. It is therefore urgent to develop novel approaches for reliable detection of neural stem cell-like cells in GBM. Here we report a luminescent conjugated oligothiophene (LCO), named GlioStem (p-HTMI), for non-invasive and non-amplified real-time detection of live human patient-derived GBM cells and embryonic neural stem/progenitor cells (NSPCs). Within a maximum of 10 minutes after administration of the molecule in vitro, in the existing media, fluorescence emission was observed without any modulation of the cells or additional vehicle, resulting in efficient detection of cytoplasmic luminescent signal in NSPCs or GBM cells from rodents and humans, detectable at Alexa488/GFP wavelength. GlioStem is functionalized with a methylated imidazole moiety resembling the side chain of histidine/histamine, and non-methylated analogues were not functional. In vitro, GlioStem was shown to identify fetal cortical NSPCs from rat (FGF2-expanded), embryonic stem cell-derived NSPCs from mouse (FGF2/EGF-expanded), and FGF2-exposed C6 glioma cell cultures from rat, but not any other cell types investigated. Cell sorting experiments of patient-derived, FGF2/EGF-expanded GBM cells demonstrated that GlioStem in addition to NSPC-markers like Nestin and Sox2 labeled the same population (overlap &gt; 90%) of cells as CD271, a proposed marker for stem cell-like cells and rapidly migrating cells in glioblastoma. Our results suggest that the LCO GlioStem is a versatile tool for immediate and selective detection of subpopulations of neural stem and glioma cells.
STEM-22. EFFECT OF A SRC INHIBITORY PEPTIDE BASED ON CONNEXIN43 ON NEURAL STEM CELLS WITH GLIOMA-DRIVER MUTATIONS
Authors: Álvarez-Vázquez Andrea , San-Segundo Laura , Cerveró-García Pilar , Flores-Hernández Raquel , Ollauri-Ibáñez Claudia , Segura-Collar Berta , Sánchez-Gómez Pilar , Pollard Steven M , Tabernero Arantxa 
Published: November 2022

Abstract
Glioblastomas are one of the most malignant tumors. Glioma Stem Cells (GSCs) are a subpopulation of cells resistant to standard treatments, responsible for tumor recurrence. Strong evidence supports that Neural Stem Cells (NSCs) from the subventricular zone (SVZ) are the origin of GSCs. This transition frequently concurs with epidermal growth factor receptor (EGFR) overexpression or mutations, such as EGFRvIII. Our group designed a cell penetrating peptide based on connexin43 (TAT-Cx43266-283) that inhibits the oncoprotein c-Src and therefore targets GSCs, increasing survival in glioma-bearing mice. Because c-Src and EGFR signaling are closely related, we explored the effect of TAT-Cx43266-283 in the transition of NSCs to GSCs. We compared the effect of TAT-Cx43266-283, control peptides, temozolomide and erlotinib, a common EGFR inhibitor, in SVZ NSCs (Control-NSCs) and murine NSC and patient-derived GSCs with key glioma-driver mutations in Nf1, PTEN and EGFR. EGFR signaling pathway was analyzed by Western blot. Furthermore, we are analyzing the effect of TAT-Cx43266-283 in a mouse model where tumors originate from SVZ NSCs with glioma-driver mutations. Our results showed a strong effect of TAT-Cx43266-283 in cell viability of NSCs and patient-derived GSCs with EGFR amplification or active EGFRvIII, without significant effects in Control-NSCs. Importantly, we found a lower effect of temozolomide, erlotinib, and other control peptides in these cells. Western blot analyses suggest that TAT-Cx43266-283 decreases EGFR, EGFRvIII and c-Src activity. Preliminary in vivo results suggest that TAT-Cx43266-283 decreases tumor size and enhances survival in mice bearing gliomas initiated by altered NSCs. So far, our results show that TAT-Cx43266-283 impairs EGFR signaling pathway with the subsequent reduction in the proliferation and survival of NSCs or GSCs with EGFR alterations. These results stress the relevance of TAT-Cx43266-283 as a future therapy against glioblastoma, alone or in combination with other treatments.
STEM-26. ASTROCYTIC SOX2+ CELLS RELAY IN NON-CANONICAL GLI SIGNALING TO FACILITATE MEDULLOBLASTOMA RELAPSE
Authors: Swiderska-Syn Marzena , Mir-Pedrol Julia , Oles Alexander , Babcock Benjamin , Martin Vanesa , Krieg Carsten , Gershon Timothy , McCrea Heather , Rodriguez-Blanco Jezabel 
Published: November 2022

Abstract
Medulloblastoma is the most common malignant pediatric brain tumor. Despite the fair efficacy of current treatment approaches, 30 % of patients with medulloblastoma relapse. Cells expressing the stemness biomarker SRY (sex determining region Y)-box 2 (SOX2) are known to play key roles in sustaining medulloblastoma growth, providing chemo-resistance, and driving tumor relapse. It is therefore critical to elucidate the underlying mechanisms that propagate these cells and ensure therapies to target them. Single cell sequencing analyses revealed the existence of a subset of astrocyte-like SOX2+ cells expressing biomarkers indicative of SHH signaling activation. Intriguingly, such SOX2+ cells were not affected by the upstream SHH inhibitor vismodegib. Using SOX2+ enriched cultures, we observed that astrocyte-like SOX2 cells not only express SHH effectors, but require GLI signaling to proliferate, and that GLI is activated in a non-canonical and MYC dependent manner. Importantly, in vivo inhibition of SHH signaling downstream of SMO depleted the vismodegib resistant SOX2+ cell pool, while reduced the ability of residual medulloblastoma tissues to engraft in vivo. Our data show that in medulloblastoma, a subset of SOX2+ tumor cells rely on non-canonical GLI signaling to propagate, and emphasizes the importance of using therapies that deplete SOX2+ cells to prevent tumor recurrence.
CCRG-03. STUDY OF PIWI-INTERACTING RNAS IN GLIOBLASTOMA PATHOLOGY: A NEW LEVEL OF REGULATION OF GLIOBLASTOMA STEM CELLS?
Authors: Fadrus Pavel , Vybihal Vaclav , Siegl Frantisek , Smrcka Martin , Slaby Ondrej , Sana Jiri 
Published: November 2022

Abstract

BACKGROUND
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 then play a key role in their biology, particularly in germ and stem cells. A number of piRNA molecules are also promising biomarkers useful for prognosis and prediction of disease progression.Material and

METHODS
Glioblastoma stem cells 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 differentiated cells. RNA was isolated from the selected paired primocultures and subsequently checked for quality by capillary electrophoresis. NEBNext Small RNA Library Prep Set was used for the preparation of cDNA libraries. Sequencing analysis was performed using the Next500/550 High Output v2 Kit for 75 cycles and the NextSeq500 sequencer.

RESULTS
Twenty paired primary cultures were prepared to verify the expression of CD133 and SOX2 in GSCs, the ability to establish tumors in immunodeficient mice, and the ability to differentiate. At this time, we finished sequencing of piRNA molecules and acquired data are bioinformatically analysed. Subsequently, we will perform analysis of selected piRNAs to be diagnostic and prognostic biomarkers on the GBM samples and non-tumor brain controls. All results will be presented at the conference.

CONCLUSION
Based on recent studies, it is evident that piRNA expression can have a significant impact on GBM biology. Their expression within GSCs may then provide a tool for identifying and targeting these cells and could be usable as a diagnostic and prognostic biomarker in 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, NV19-03-00559, Conceptual development of research organization FNBr, 65269705
CCRG-04. TARGETING THE CELL-CYCLE EFFECTS OF TUMOR TREATING FIELDS (TTFIELDS) TO INCREASE ITS EFFICACY
Authors: Slangen Paul , Elbakary Basma , van Mil Lotte , de Gooijer Mark , Krenning Lenno , van den Broek Bram , Mertz Marjolijn , Van Tellingen Olaf , Borst Gerben 
Published: November 2022

Abstract
Tumor Treating Fields (TTFields) are a novel, non-invasive FDA-approved treatment modality for glioblastoma (GBM) that utilises alternating electric fields. Our research aims to elucidate the effect of TTFields on the cell cycle to advance the understanding of TTFields and find novel targets for increasing its efficacy. We studied the cell cycle progression using live-cell imaging (inovitro Live™) of PIP-FUCCI-transduced GBM cells, EdU incorporation, RPA foci and fiber assay (in U251-MG, SNB-19, A172). These tools allow us to track individual cells evaluate the time spent in each cell cycle phase, and study the ultimate cell fate in control and treatment conditions concerning the cell cycle and replication fork dynamics. Statistical testing was performed by 2-way ANOVA, Tukey-corrected for multiple comparisons, and Kruskal-Wallis test. TTFields causes an immediate delay in S and G2 cell cycle phase, with a subsequent G1 arrest. Wee1 inhibition by AZD1775 forces cells through S-phase and abrogates the subsequent TTFields-induced G2-checkpoint, preventing DNA repair. Combined treatment with TTFields and AZD1775 led to decreased incorporation of EdU and an increase in S-phase-specific phospho-RPA foci. TTFields reduced significantly the DNA fiber length after 12, 24, 48 and 72 hrs of exposure (10.2, 8.3, 7.1 and 5.6 vs 13 um), with also an increase in replication fork asymmetry at these time points (0.56, 0.45, 0.49, and 0.47 vs 0.9 ratio) respectively. CombiningTTFields with either Wee1, ATR, or Chk1 inhibition synergistically decreased clonogenic survival. TTFields induced cell cycle effects with the induction of replication stress, which is aggravated by inhibition of Wee1. Additionally, Wee1, ATR, and Chk1 inhibition dramatically boost the efficacy of TTFields in vitro. This combination strategy has promising clinical potential and will be tested in vivo in different GBM xenografts.
Cell signaling and signaling pathways
CSIG-04. BMP4 INDUCTION OF A SENESCENCE-LIKE PHENOTYPE IN HUMAN GLIOBLASTOMA CELLS IS PARTIALLY DEPENDENT ON P21
Authors: Niklasson Mia , Dalmo Erika , Westermark Bengt 
Published: November 2022

Abstract
Bone morphogenetic protein 4 (BMP4) was initially suggested as a potential differentiation-inducing factor to be used in the therapy of glioblastoma. We and others have however demonstrated that the response is reversible, variable among patient samples, and heterogeneous within the same cell line. To deepen our knowledge on how BMP4 affects different types of glioblastoma cells, we treated phenotypically different clones from the same patient tumor—a multitherapy-sensitive/proneural-like (SENS/PN) clone and a multitherapy-resistant/mesenchymal-like (RES/MES) clone—with BMP4. In the SENS/PN clone, BMP4 turned on a mesenchymal-related gene program, whereas this response was less prominent in the RES/MES clone. Untreated SENS/PN cells were smaller than RES/MES cells, but both clones responded to BMP4 by cell size enlargement. Increase in cell size has been suggested to precede senescence; young cells are smaller than old cells that eventually enter replicative senescence. BMP4 induced a senescence-like phenotype in a subpopulation of cells, demonstrated by induction of senescence-associated (SA)-β-gal, p21 up-regulation, lamin B1 down-regulation, as well as increased lysosomal mass and granularity. This was more pronounced in the RES/MES clone than in the SENS/PN clone, and it was dependent on canonical SMAD signaling. Senolytic treatment ablated the SA-β-gal positive cells and reduced the p21 level at the population level. Targeted deletion of p21 abolished BMP4-induced SA-β-gal and increase in cell growth, while lamin B1 down-regulation remained, demonstrating that p21 signaling is crucial for one part of the senescence induction by BMP4. We are currently further investigating a connection between cell size, mesenchymality and senescence. We hypothesize that large mesenchymal-like cells are closer to senescence than smaller proneural cells within the cell culture. A combination of senescence-induction and senolytic treatment may open treatment opportunities to target therapy-resistant glioblastoma cells.
CSIG-07. GAIN-OF-FUNCTION MUTANT P53 REGULATES LONG-NONCODING RNAS IN GLIOBLASTOMA
Authors: Zhang Ying , Yuan Feng , Grello Cassandra , Reon Brian , Gibert Myron , Dube Collin , Dutta Anindya , Holland Eric , Abounader Roger 
Published: November 2022

Abstract
P53 is frequently mutated in most human cancers, including glioblastoma (GBM). Many p53 mutants acquire gain-of-function oncogenic effects through only partially understood mechanisms. To investigate the role of gain-of-function mutant p53 (MUT-p53) in GBM, we performed ChIP-seq of wildtype p53 (WT-p53) and MUT-p53 GBM cell lines. Among 2834 unique peaks reads in MUT-p53 cells, we found 242 long non-coding RNAs (lncRNAs) with up to 145 fold enrichment relative to WT-p53. LncRNAs regulate many molecular and cellular functions, including gene expression, cell proliferation, death, cancer stem cell renewal and differentiation. We selected lncRNAs SOX21-AS1 and LINC00643 with highly enriched binding by MUT-p53 and investigated their expressions and functions in the p53 pathway. We performed ChIP confirmation of MUT-p53 binding to the promoters of these lncRNAs. We found that these lncRNAs are deregulated in GBM and correlated with GBM patient survival in the TCGA database. To investigate the functions of these LncRNAs, we knocked down their expressions by siRNA, and found significant cell death induced by si-SOX21-AS1, but not by si-LINC00643. Overexpression of LINC00643 in GBM cells led to inhibition of GBM cell proliferation, migration, invasion and in vivo xenograft growth. LINC00643 mediated the effects of MUT-p53. Co-expression of human LINC00643 and its mouse homologous in a RCAS transgenic mouse model of GBM reduced tumor growth and improved animal survival. To elucidate the mechanisms of action of the lncRNA, we performed Chromatin Isolation by RNA purification high-throughput sequencing (CHIRP-seq) to identify its binding targets. We found that LINC00643 binds to HIF1a 5’ promoter/enhancer region. Overexpression of LINC00643 in GBM cells at hypoxia growth condition reduced HIF1a mRNA and protein expression. Our study shows for the first time that gain-of-function mutant p53 regulates a subset of lncRNAs and that the lncRNAs mediate the oncogenic effects of the MUT-p53 in GBM.
CSIG-08. MASS CYTOMETRY IDENTIFIES LOCATION-SPECIFIC TUMOR POPULATION ASSOCIATED WITH CLINICAL PROGRESSION IN GLIOBLASTOMA
Authors: Alvarado Alvaro , Bastola Soniya , Muthukrishnan Sree Deepthi , Alban Tyler , Tessema Kaleab , Nakano Ichiro , Lathia Justin , Goldman Steven , Kornblum Harley 
Published: November 2022

Abstract
Recent advances in single cell expression technology have allowed us to appreciate the complexity in tumoral heterogeneity of solid tumors such as glioblastoma (GBM), which remains uniformly fatal despite aggressive therapies. Currently, there is no knowledge about cell identities and functional characteristics of the different tumoral populations. To this end, we created a 28-marker mass cytometry panel composed of signaling markers, including phospho-proteins, and lineage markers that have been associated with GBM or are considered putative cancer stem cell markers. We evaluated lines established from core and edge areas from the same patient and validated to be functionally different with RNA expression and in vivo experiments. We found clusters shared by cells from both regions as well as region-specific ones. In order to better characterize these, we used marker enrichment modeling (MEM) and generated machine-readable labels. These were compared to MEM labels generated after using the same signaling markers to cluster primary samples from newly diagnosed or recurrent patients. Similarly, MEM labels were obtained from patients in a clinical trial treated with capecitabine that had positive or negative responses. Clusters from edge cells were found in higher proportion in recurrent tumors and were correlated with negative responses to chemotherapy. Initial analyses have also been done correlating immune clusters with tumor clusters in the same samples. Edge clusters are characterized by a CD44+ SOX2+ Nestin+ population. Treatment of tumor cells with temozolomide and irradiation results in an increase of this population in vitro. We are currently conducting single cells analyses to characterize this population as well as in vivo studies to validate the tumor-immune interactions. Our studies exploit single cell cytometry to underscore pathways that are preferentially use by different populations and affect immune cells with the goal of establishing therapeutic efforts to extend patient survival.
CSIG-12. BXQ-350 INDUCES MITOPHAGY IN GBM CELLS LEADING TO CELL DEATH
Authors: Wilkins Nikhil , Furnish Robin , Stephens Tim 
Published: November 2022

Abstract

BACKGROUND
Mitophagy is an autophagic phenomenon where defective mitochondria are selectively degraded. When mitochondria need recycling, the cell upregulates the production of the lipid C-18 ceramide on the mitochondria and lipoprotein LC3B-II on the lysosome, which transitions to an autophagolysosome. This creates a signal causing the degradation of mitochondria. Here we show that BXQ-350, a novel protein-lipid small molecule consisting of the ceramide creating protein Saposin C and the lipid Dioleoyl Phosphatidylserine (DOPS), induces glioblastoma cells into performing mitophagy. Increased production of mitophagy associated proteins, C-18, and LC3B-II leads to the degradation of healthy mitochondria followed by cell death.

METHODS
The GBM cell lines, Gli36 and U87 cells, were used to determine if increased mitophagy was occurring in response to BXQ-350 treatment. Protein abundance was measured using SILAC proteomics. Ceramide levels were analyzed using LC/MS-MS. Western Blot analysis was used to measure differences in LC3B-II levels. Mitophagy was measured using Mtphagy Dye from Dojindo and the BioTek Cytation 5 microscope.

RESULTS
Proteomics showed cells treated with BXQ-350 had an increase in proteins associated with overexpression of C-18 ceramide or autophagolysosome maturation. Western blots analysis showed a marked increase in LC3B-II in BXQ-350 treated cells. Furthermore, cells treated with BXQ-350 had increased levels of C-18 ceramide. The Mtphagy dye showed that as BXQ-350 concentrations increased, the percentage of cells positive for Mitophagy increased. To determine if mitophagy occurs in association with BXQ-350 cytotoxicity, medias with varying BXQ-350 cytotoxicity were used. The Mtphagy dye showed cytotoxic BXQ-350 medias increased levels of mitophagy, while noncytotoxic medias display no increase in mitophagy.

CONCLUSION
BXQ-350 causes the increase of mitophagy signaling proteins, C-18 ceramide and LC3B-II production, resulting in the formation of autophagolysosomes which track towards the mitochondria initiating mitophagy. This leads to aberrant mitophagy where cells degrade healthy mitochondria leading to mitophagic cell death.