bluuebunny/crossref_metadata_2025 huggingface dataset:
TMIC-48. HYPOXIC AND PERI-NECROTIC ZONES IN GLIOBLASTOMA CAUSE HIPPO-OFF SIGNALING, POTENTIALLY SUPPORTING GLIOMA STEM CELLS
Authors: Shih Ling-Kai , Li Jiabo , Markwell Steven , Carneiro-Lobo Tatiana , Olson Cheryl , Brat Daniel
Published: November 2022

Glioblastoma (GBM) is the most common and malignant primary brain tumor with pre-necrotic and post-necrotic growth phases. The development of necrosis is associated with extensive tumor micro-environmental (TME) rearrangement and rapid biologic advancement. Compared to pre-necrotic GBMs, post-necrotic GBMs are heterogenous and contain multi-focal regions of necrosis surrounded by pseudopalisading cells and characterized by severe hypoxia. Hypoxic, peri-necrotic zones are highly enriched for glioma stem cells (GSCs) and tumor associated macrophages (TAMs), the latter accounting for 30-40% total tumor cellularity. Using Ivy-GAP data, we performed gene set enrichment analysis (GSEA) for stem-cell associated markers, comparing peri-necrotic to non-necrotic regions. We also used TCGA/CGGA data to identify correlations between hypoxia markers and classic stem-cell associated markers (SOX2, SOX9, nestin, STAT3, prominin). None of these showed enrichment in the peri-necrotic zone or association with hypoxia markers. However, we uncovered strong associations of Hippo-off signaling with peri-necrotic regions, signifying activation of YAP/TAZ transcriptional activation. We therefore hypothesized that Hippo-off (YAP/TAZ transcriptional activation) in hypoxic peri-necrotic zones promotes GSC self-renewing division. To support this, we performed GSEA for Hippo pathway readout genes and found peri-necrotic enrichment of Hippo regulated genes CYR61, BIRC2, SERPINE1 and AREG. Using patient-derived GBM neurosphere cultures, we exposed glioma cells to 1% hypoxia and found that p-YAP1 and p-TAZ protein levels were downregulated compared to normoxia (21%) at 24 hrs, correlating with Hippo-on. Hippo pathway gene targets were also upregulated, indicating activation of YAP/TAZ transcription. Using Ivy-GAP, TCGA and CGGA data, we also found peri-necrotic/hypoxic enrichment of two cytokines, IL-1β and IL-6, known to trigger YAP/TAZ activation. Using cytokine multiplex analysis, we observed an upregulation in both IL-1β and IL-6 secretion from hypoxic monocytes and microglia, raising the possibility that these cytokines directly stimulate Hippo-off and promote GSC self-renewing division in the GBM peri-necrotic niche.
TMIC-51. SEX-SPECIFIC T CELL BEHAVIOR DRIVES DIFFERENTIAL IMMUNE RESPONSES IN GLIOBLASTOMA
Authors: Lee Juyeun , Sneiderman Chaim , Kohanbash Gary , Sangwan Naseer , Lathia Justin
Published: November 2022

Glioblastoma (GBM) is the most common primary malignant brain tumor and shows poor outcomes as a median survival is 12-18 months with current standard-of-care of therapy. GBM exhibits sex differences in incidence and overall survival, with males experiencing a higher incidence and a worse prognosis compared to females. However, the role of immune cells in GBM sex differences outside of myeloid cells remains poorly understood. Using a syngeneic mouse GBM model, we recapitulated the sex differences observed in patients, with shortened survival in male hosts compared to female hosts. These findings were not recapitulated in immuno-deficient mouse strains such as NSG and RAG1KO mice, suggesting a role for immune system, specifically T cells, in GBM sex differences. Flow cytometry analysis of tumor-infiltrating leukocytes revealed that more T cells were found in female tumors, whereas male tumors were enriched in macrophages. Additionally, more T cells in male tumors exhibited high levels of inhibitory receptors, whereas most female T cells were more functional as measured by expression of anti-tumor cytokines such as TNF, IFN-gamma, and granzyme B. A bone marrow chimera model (BMC) revealed that male T cells retained their phenotype in female hosts, whereas female T cell behavior was affected by the male environment. Yet survival analysis on BMC model and adoptive transfer model suggests strong immune cell-intrinsic effect on controlling tumor progression, further supported by in vitro T cell exhaustion model. Lastly, we found that males have more progenitor exhausted T cells, which led to better response to anti-PD1 treatment. Collectively, these results suggest that both cell-intrinsic and cell-extrinsic factors regulate T cell activity in a sex-specific manner, providing insights to develop sex-specific therapeutic approaches.
TMIC-54. THE ROLE OF TUMOR MICROENVIRONMENT DERIVED GROWTH FACTORS IN PEDIATRIC BRAIN TUMORS
Authors: Cruz Andrea , Locke Abigail , Halligan Katharine , Sanders Lauren , Cheney Allison , Stanton Ann-Catherine Jean , Koncar Robert , Broniscer Alberto , Vaske Olena M , Pearce Thomas , Marker Daniel , Wiley Clayton , Mack Stephen C , Filbin Mariella , Pollack Ian F , Agnihotri Sameer
Published: November 2022

BACKGROUND

High-grade gliomas (HGGs) are the most common fatal intrinsic brain tumors in pediatric patients. H3K27-altered diffuse midline gliomas (H3K27-DMGs), a subgroup of HGGs defined by a histone 3 position 27 alteration, are especially aggressive and result in the poorest patient outcomes. Despite in-depth genomic characterization, the 5-year survival rate has yet to improve beyond 2% following diagnosis. A common feature of H3K27-DMGs is infiltration of microglia, macrophages, other myeloid cells, collectively referred to as GAMs, and a small population of T-cells. The contribution of non-tumor cells in the tumor microenvironment (TME) can both promote and or inhibit tumor growth, thus representing an opportunity in the pursuit of novel therapeutics. Using bioinformatic analysis on a human H3K37-DMG single cell-RNA sequencing dataset, we reveal several cell-to-cell communication signaling networks, mediated by ligand and receptor pairs, between GAMs and tumor cells, respectively.

HYPOTHESIS

Microglial-derived growth factors activate oncogenic signaling pathways via paracrine signaling axes, thus promoting H3K27-DMG tumor cell proliferation and growth.

METHODS

I will validate these findings and test their therapeutic potential using co-culture studies, CRISPR and shRNA gene silencing, and phospho-proteomics technology.

RELEVANCE

This research provides further insights on the contribution of non-tumor cells in the TME towards H3K27-DMG cell proliferation and growth and could potentially inform future therapy paradigms.
TMIC-81. INCREASED LEVEL OF IGFBP2 PROMOTES TUMOR METASTASIS IN SHH MEDULLOBLASTOMA
Authors: Kunhiraman Haritha , McSwain Leon , Shahab Shubin , Kenney Anna
Published: November 2022

Medulloblastoma (MB) is the most common pediatric brain malignancy. MB comprises 5 major subgroups known as WNT, SHH p53wt, SHH p53mut, Group 3 and Group 4. Among the four MB subgroups SHH group is the most dominant molecular subgroup in infants and adults. These tumors are proposed to arise from cerebellar granule neuron precursors (CGNPs), whose developmental expansion requires SHH signaling from the neighboring Purkinje neurons. Previous reports suggest that SHH group features a unique tumor microenvironment compared with other MB groups. To better understand how SHH MB cells interact with Tumor Microenvironment, we performed cytokine array analysis of culture media from SHH group Patient Tumor cells, spontaneous SHH MB mouse tumor cells and SHH MB cell lines. Further, confirmed these results using ELISA, Western blot, and immunofluorescence from human SHH MB cell lines, Smo/A1 mouse tumor primary cells and PZp53Med cell lines. In continuation to the observation of IGFBP2 expression in various cell types in single cell analysis, we analyzed the presence of IGFBP2 in astrocytes using Smo/A1 mouse tumor Immunohistochemistry. Our data showed increased levels of IGFBP2 produced by SHH MB cell lines compared to others. We analyzed the role of IGFBP2 in SHH MB tumor growth and metastasis. IGFBP2 knock-down stable cell lines showed phenotypic changes including reduced cell proliferation, cell migration and EMT. Further western blot analysis of IGFBP2 KD cells showed reduced expression of EMT markers also reduced the activation of STAT3. Our preliminary in vitro data suggest IGFBP2 exerts it metastasis-promoting role in SHH MB by regulating the expression of EMT marker proteins and matrix remodeling proteins. Further functional studies suggest that in SHH MB, IGFBP2 may regulate a STAT3-mediated EMT program to metastasize. These findings provide a strong rationale for further pursuing how IGFBP2 promotes medulloblastoma tumor cell growth and migration in vivo.
TMIC-82. LIPOCALIN-2 MEDIATES CELL-INTRINSIC AND CELL-EXTRINSIC FUNCTIONS IN GLIOBLASTOMA
Authors: Wang Sabrina , Silver Daniel , Troike Katie , Deleyrolle Loic , Lathia Justin
Published: November 2022

Glioblastoma (GBM) is the most common primary brain tumor with a median survival of 17-20 months. Despite therapeutic treatments including surgery, radiation, chemotherapy, a subset of GBM clones, termed cancer stem cells (CSCs) are radio-resistant and chemo-resistant, leading to mortality of the patient. CSCs also have altered metabolic profiles and have an enhanced capacity to scavenge nutrients from their microenvironment, including iron. Lipocalin-2 (LCN2) functions to sequester iron and is traditionally considered an inflammatory marker through its function of limiting iron for bacterial usage. LCN2 has been described to have both a pro and anti-tumorigenic and has context-dependent functions depending on iron status. LCN2 has been shown to be important in brain metastasis, but its role in GBM is still largely unknown. To investigate how LCN2 plays a role in the GBM microenvironment, we orthotopically implanted syngeneic mouse GBM cells into male and female LCN2 knockout and wild-type mice. Female LCN2 knockout mice succumbed to tumors faster compared to males, revealing another example of sex differences in the tumor microenvironment. To assess a cell-intrinsic function for LNC2, we added recombinant LCN2 to mouse GBM cell lines and saw that proliferation also increased. When we probed for receptor expression in our knockdown cell lines, we saw that the LCN2 canonical receptor, SLC22a17, was upregulated in response. Furthermore, in a subset of slow-cycling cells of CSCs, LCN2 was shown to be up-regulated. Taken together, these data suggest that LCN2 functions in an iron-dependent manner to affect proliferation and sex-specific tumorigenesis. Given the fact that males have more iron than females, it is worth investigating the role of iron in GBM sex difference progression and therapeutic targets.
CNSC-32. ACTIN SUBUNIT STRUCTURAL DIVERSITY IN SHH MEDULLOBLASTOMA: IMPLICATIONS FOR CANCER BIOLOGY AND NEURODEVELOPMENT
Authors: Beaulieu Jamie , Knapp David , Picard Daniel , Remke Marc , Sadikot Abbas , Diaz Roberto
Published: November 2022

BACKGROUND

Altered expression of actin subunits is found in multiple cancers. Alpha-cardiac actin (ACTC1) is expressed in sonic hedgehog medulloblastoma (SHH MB) and regulates both cell survival and migration. We hypothesized that cardiac actin has a unique structural and functional role in SHH MB and may be expressed in post-natal granule cell progenitors (GCPs) which share similar gene expression profiles.

METHODS

Actin subunit protein expression (ACTC1, ACTB, ACTG1, and ACTA2) was evaluated by mRNA expression profiling within established datasets and across MB cell lines by Western blot (WB). Immunofluorescence microscopy was performed to assess intracellular actin subunit localization. The composition of each subunit in filamentous (F-) actin was evaluated. Furthermore immunoprecipitation of filamentous ACTC1 was performed to assess protein-protein interactions. The ratio of F-actin to globular (G-) actin was determined by WB in SHH MB cells exposed to mitotic inhibitor. Cell viability was determined by colony formation following over-expression of dominant-negative mutant ACTC1 (P116C & G247D).

RESULTS

ACTC1, ACTG1, and ACTB are the predominant actin subunits in SHH MB. Immunofluorescence microscopy demonstrated co-localization of ACTC1 with ACTG1 and ACTB but not ACTA2. ACTC1 showed a unique cortical localization pattern. Post-natal MATH1 positive mouse GCPs (P3-5) demonstrate similar distribution of ACTC1 in cortical F-actin. WB analysis of ACTC1 pulldown from SHH MB cell F-actin fractions showed co-precipitation with ACTG1 and ACTB but not ACTA2. ACTC1 F-actin to G-actin ratio is maintained in cells that show resistance to mitotic inhibition. Expression of mutant ACTC1 G247D resulted in reduced cell survival.

CONCLUSIONS

ACTC1 subunit is incorporated into F-actin in SHH MB and GCPs. Filamentous ACTC1 forms a protein complex with ACTG1 and ACTB. Disruption of ACTC1 polymerization results in reduced cell survival. These findings suggest a functional role of ACTC1 in both SHH MB and in GCPs which are important in cerebellar development.
MODL-07. Sensitivity of Tumor Treating Fields (TTFields) treatment planning to tumor segmentation errors
Authors: Ze’evi Oshrit , Marciano Tal , Naveh Ariel
Published: November 2022

Tumor Treating Fields (TTFields) are electric fields that disrupt cellular processes critical for cancer cell viability and tumor progression. TTFields are administered through two pairs of capacitive arrays placed on the patient’s scalp. The effect of TTFields has been shown to be dose dependent. An in-silico study based on imaging and survival data from the phase 3 EF-14 study demonstrated a correlation between simulated field intensity and overall survival. Novocure is developing a simulation-based patient-specific treatment planning system aimed towards maximizing TTFields dose delivered to the patient’s tumor. Different options are presented to the treating physician that are ranked based on dose. To create patient-specific planning, physicians are required to segment the pathological tissues on a T1-weighted MRI. These tissues may include the enhancing tumors, resection cavity and necrotic core. Segmentation of medical images is an error-prone procedure. Such errors may influence the calculation of expected TTFields dose delivered to the tumors. In this work, we examined how different segmentations of the tumors affect the delivered dose, by placing mock tumors of different compositions within a simplified ellipsoidal model. Simulations were then used to assess field distributions in these tumors. Tumors of different enhanced volumes were then compared by LMiPD values. Our results show that 10% differences in the segmented enhancement result in less than 5% relative error in LMiPD. To enforce the validity of the simplified model, we performed the same analysis for two patient specific models. In both cases, the relative difference in segmentation was about 5%, and consequent errors in LMiPD was <5% as well. In conclusion, when using a simulation-based TTFields treatment planning system, small differences in the segmentation of the enhancing component of mock glioblastoma tumors in both simplified ellipsoid and patient specific models lead to small differences in LMiPD.
MODL-10. SYSTEMATIC CHARACTERIZATION OF GLIOBLASTOMA GROWTH PATTERNS REVEALS INVASION ROUTE-SPECIFIC FUNCTIONAL SIGNATURES AND DRUG VULNERABILITIES
Authors: Krona Cecilia , Rosén Emil , Kundu Soumi , Olausson Karl Holmberg , Larsson Ida , Elfineh Ludmila , Doroszko Milena , Elgendy Ramy , Wikström Johan , Jörnsten Rebecka , Nelander Sven
Published: November 2022

Glioblastomas (GBMs) are malignant brain tumors characterized by uncontrolled, invasive growth along multiple anatomical pathways. GBM is well-studied on a genetic and molecular level, but clinically relevant, experimentally tractable models of invasive growth are largely lacking. We report an integrated study of patient-matched information including clinical and radiological data, patient-derived xenografts, genomics, transcriptomics, epigenomics, and drug response data. Our hypothesis is that joint variation across these levels can expose treatments and biomarkers associated with glioblastoma invasion and recurrence. In total, 64 patient-derived cell lines (PDCLs) were injected into the striatum of n >= 4 mice each, of which 45 PDCLs successfully formed tumors. The tumor-forming PDCLs were each scored with respect to 10 distinct growth characteristics (n= 182 mice). The repertoire of phenotypes was highly divergent and included clear cases of perivascular invasion, white matter invasion, perineuronal satellitosis, and gliosarcoma. We explored if cellular pathways, monitored by RNA sequencing, could account for these differences. DNA repair, Wnt-, and Notch-pathways were predictive of tumor initiation, whereas pathways related to hypoxia, metabolic reprogramming, cellular stress, and inflammation reduced tumor initiation capacity of PDCLs. Transcriptional signatures were also strongly predictive of route-specific invasion. Diffuse invasion was predominantly seen in classical-subtype PDCLs with astrocytic or outer radial glia-like signatures. Proneural PDCLs, in turn, grew as solid tumors with an invasive peripheral region, and mesenchymal tumors were demarcated and invaded around vasculature. To explore the therapeutic implications of our findings, we used our data-driven method TargetTranslator to predict the drug vulnerabilities of different types of invasive glioblastoma. Transcriptional signatures of diffusively growing tumors suggested a vulnerability to neuroactive substrates while perivascular invading tumors would be more susceptible to CDK- and RTK inhibitors. We expect that focusing on drivers of these functional signatures will greatly facilitate the search for effective compounds targeting distinct growth behaviors.
MODL-11. NOVEL IMMUNOTHERAPY MODEL IN AGGRESSIVE PEDIATRIC H3K27M MUTANT GLIOMAS WITH INDUCIBLE TGFBR2 AND LAG3 EXPRESSION
Authors: Ljubimov Vladimir , Linesch Paul , Sabet Sara , Krutikova Viktoria , Mehdikhani Shaunt , Pacheco David Rincon Fernandez , Danielpour Moise , Breunig Joshua
Published: November 2022

INTRODUCTION

Gliomas are considered immunologically “cold” tumors that mask themselves from the immune system. Immunotherapy, a breakthrough for cancer treatment, remains inefficient for gliomas. H3K27M mutant diffuse midline glioma has poor survival. Using our innovative mosaic analysis with dual recombinases technology (Cell 2019), we further created a novel murine model of H3K27M glioma with an inducible expression of LAG3 and TGFBR2 to make the tumor recognizable by the immune system. This allows studying molecular mechanisms of “perfect” immunotherapy in pediatric gliomas with histone H3.3 mutations.

METHODS

To make a novel murine model, H3K27M plasmids with doxycycline-inducible expression for TGFBR2 and LAG3 were designed, sequenced, and replicated. For in vitro validation, murine neuronal stem cells were nucleofected with these plasmids, grown and sorted. Doxycycline was added to the experimental group in vitro to induce expression. The plasmids were then electroporated into neonatal MGMT mice (n=21) of both genders. Control mice were used on a standard diet (n=9) and experimental mice (n=12) were given doxycycline in food to induce TGFBR2 and LAG3 expression in vivo. Mice were followed up for survival.

RESULTS

Plasmid sequencing validation demonstrated their high purity. In vitro nucleofected neuronal stem cells showed significant increase in TGFBR2 and LAG3 expression by immunofluorescent staining after addition of doxycycline. The mouse survival study showed a significant benefit with treatment group surviving 46% longer (135 ± 41.9 days vs. 92.6 ± 32.7 days in the control group, p=0.030). Conclusion and Future Directions: We have successfully modified H3K27M glioma to have inducible expression of TGFBR2 and LAG3 to simulate “perfect” immunotherapy. The survival study showed a significant survival benefit. The development of organoid transplants for more uniform testing and next-generation sequencing is ongoing. This can lead to the emergence of novel immune therapies and uncover novel mechanisms of tumor response to treatment.
MODL-17. SEGMENTATION OF PRE AND POST-TREATMENT GLIOMA TISSUE TYPES INCLUDING RESECTION CAVITIES
Authors: White Nathan , Baig Saif , Vidic Igor , Mastorakos George , Smith Robert , Dale Anders , McDonald Carrie , Beaumont Thomas , Seibert Tyler , Peddi Srinivas , Hattangadi-Gluth Jona , Farid Nikdokht , Kesari Santosh , Rudie Jeffrey
Published: November 2022

PURPOSE

Measuring gliomas is a time-intensive process with significant inter-rater variability in post-surgical residual tumor and resection cavities. This likely contributes to the delayed assessment of progression and in-field recurrences from radiation. Automated segmentation of pre and post-treatment gliomas could reduce inter-rater variability and increase workflow efficiency for routine longitudinal radiographic assessment and treatment planning. We evaluated whether a 3D neural network is comparable to expert assessment of pre and post-treatment diffuse gliomas tissue types and resection cavities.

METHODS

A retrospective cohort of 647 MRIs of patients with diffuse gliomas (average 55.1 years, 45% female, 396 pre-treatment and 251 post-treatment, median 237 days post-surgery) from The Cancer Imaging Archive were stratified by operation status and tumor grade and randomly split into training (536) and testing (111) samples. T1, T1-post-contrast, T2, and FLAIR images were registered, skull-stripped, and interpolated to 1x1x1. Four classes — edema/infiltrative/post-treatment changes (ED), enhancing tissue (ET), necrotic core (NCR), and resection cavities (RC) — were manually segmented by an expert neuroradiologist. Using these segmentations and the four input image modalities, a nnU-Net, a state-of-the-art 3D U-Net convolutional neural network, was trained to predict segmentations of the four classes.

RESULTS

Segmentation performance in the test set for the ED, ET, and RC classes was at the level of inter-rater reliability with median Dice scores ranging from 0.85 to 1 and relative volume errors ranging from -0.05 to 0.02. There was a near-perfect correlation between manually segmented and predicted total lesion volumes (r2 values ranging from 0.95 to 0.98 among classes).

CONCLUSIONS

Accurate, automated volumetric quantification of diffuse glioma tissue volumes may improve response assessment in clinical trials and reduce provider burden and errors in measurement.
MODL-23. MULTISCALE MODELS OF GLIOMA PROGRESSION: INSIGHTS ABOUT VASCULATURE AND THERAPY EFFECTS
Authors: Conte Martina , Surulescu Christina , Dzierma Yvonne , Knobe Sven
Published: November 2022

Gliomas are the most prevalent, aggressive, and invasive subtype of primary brain tumors, characterized by fast cell growth, strong invasion capability, and well-developed tumor vasculature. Although the research advances and the clinical trials testing the efficacy of novel combined therapies have allowed significant progress in the comprehension and treatment of gliomas, these tumors are characterized by a poor prognosis, and recurrence remains the main cause of mortality. The growth and migration of tumor cells inside the brain is a highly complex phenomenon, influenced by a multitude of intrinsic and extrinsic factors at different spatial and temporal scales. The high complexity of this invasion process remains a challenge to face and several important questions are still unanswered. In this context, mathematical models emerge as powerful tools to face these challenges. Here, we propose a multiscale approach for the description of glioma progression under the influence of vascularization. Precisely, we firstly focus on the effects of (hypoxia-driven) acidity and phenotypic heterogeneity on tumor progression. Then, we extend the setting to include vascular endothelial growth factor evolution and compare various therapeutic approaches. Starting from a microscopic description of the interactions between specific cell membrane receptors and the extracellular microenvironment, we define two different coupled systems for endothelial and tumor cell evolution. The analysis of these systems allows us to assess tumor growth and spread on the basis of DTI and glioma patient data. In particular, we perform several data-driven numerical tests aimed at assessing the role of vasculature and phenotypic heterogeneity on tumor progression and comparing the effect of different combinations of radio-, chemo-, and anti-angiogenic therapy on it.
STEM-05. GLIOBLASTOMA NEUROSPHERES DISPLAY STEMNESS CHARACTERISTICS AND UPREGULATION OF THE VEGF-A PATHWAY
Authors: Mozaffari Khashayar , Chandra Neha , Aguirre Brian , Cherian Nina , Chandla Anubhav , Wadehra Madhuri , Yang Isaac
Published: November 2022

INTRODUCTION

Glioma stem cells (GSC) constitute a subpopulation of tumor cells with stem-cell like properties and are believed to enhance glioblastoma’s ability to grow and confer resistance against chemoradiotherapy. It has been shown that non-adherent three-dimensional (3D) growth conditions facilitate neurosphere formation and enable isolation of GSCs in vitro. This can serve as an invaluable tool to better understand the role of GSCs in glioblastoma pathogenesis.

OBJECTIVE

We aimed to evaluate differentially expressed genes in non-adherent versus adherent conditions using primary tumor glioblastoma cell lines; and to identify alterations in various signaling pathways that are paramount to their growth and maintenance. Furthermore, we targeted the EMP2-HIF1α axis, a proposed regulator of VEGF expression, to determine its effects on cell viability.

METHODS

Three primary tumor glioblastoma cell lines were generated and cultured under adherent or non-adherent growth conditions. Sequencing was performed on the Illumina HiSeq3000 system, and gene counts were normalized by TMM. A pair-wise statistical analysis of over 20,000 genes was performed between the two conditions. A p-value < 0.05 was considered statistically significant.

RESULTS

After 72 hours of growth under 3D conditions, primary glioblastoma cells show an increase in stem-like properties, measured via an upregulation of SOX2 and nestin. RNA sequencing demonstrated a significant increase in expression of the following transcripts in 3D conditions compared to those grown on adherent plates: VEGF-A, EGFR, PDGFR-A, FGFR3, and TGFB2. Treatment with anti-EMP2 monoclonal antibodies significantly reduced cellular proliferation.

CONCLUSION

Our results demonstrate that neurospheres upregulate growth factor pathways to enable their survival in 3D conditions, suggesting an inherently different genetic landscape between differentiated tumor cells and GSCs. Future studies will investigate the cellular mechanisms responsible for upregulation of these genes; and to ultimately design more effective targeted therapeutic agents.
STEM-12. DISCOVERY THE ORIGIN-CELLS FOR HUMAN GLIOBLASTOMA GENESIS IN SUBVENTRICULAR ZONE
Authors: Yoon Seon-Jin , Choi Ran Joo , Oh Yoojung , Cho Se-Young , Kim Hyun Jung , Lee Joo Ho , Yeo Eunchae , Yoo Jihwan , Moon Ju Hyung , Kim Eui Hyun , Kim Se Hoon , Kim Huyn Seok , Chang Jong Hee , Kim Sangwoo , Kim Hoon , Lee Jeong Ho , Kang Seok-Gu
Published: November 2022

Human glioblastoma (GBM), originating from the subventricular zone (SVZ), occurs due to molecular disruptions in chromosomes. Most GBM tissues exhibit definitive chromosomal patterns: copy-number-variations (CNV) in chromosome 7 (gain) and 10 (loss), known as the earliest molecular events. Herein, we hypothesised that the origin-cells in SVZ of GBM patients can provide clues regarding these chromosomal alterations. We compared bulk RNA sequencing (RNAseq) data of GBM tumor tissue (n=126), tumour free GBM SVZ (n=40), and tumor-free control SVZ of non-glial tumor (n=9). Paired single-cell-level RNAseq samples of tumor free GBM SVZ (n=7) and GBM tumor tissue (n=10), were done to see cell specific CNVs. Using human SVZ and GBM samples as a background, we generated origin-cell and origin-cell-derived tumour cell using CRISPR/Cas9. In this work, we identified two GBM-origin-cell types with stem-cell signatures during single-cell level analyses of 60 SVZ tissue samples obtained from tumor-free regions of GBM patients. Furthermore, single-cell level analysis revealed that two origin-cell types in SVZ harbor ongoing patterns of CNV alterations. Among the origin-cells found in the SVZ of GBM patients, NO-like cells showed neural progenitor plus oligodendrocyte progenitor (NO) signature, while AN-like cells showed astrocyte plus neural stem cell (AN) signature. For the interconnectedness, we subjected single-cell-level RNA-seq data to ligand-receptor connection analysis. In the stem cell mode, NO-like cells was connected to AN-like cells in SVZ samples and while in the tumor samples, cycling cell was connected to AN-like cells. NO-like cells was common in TERT promoter wildtype GBM and AN-like cells was more common in TERT promoter mutant GBM. CRISPR/Cas9 models revealed accumulation copy-number alterations from non-tumorigenic origin-cells to tumor cells. These two origin-cells (NO-like cells and AN-like cells) derived from the SVZ of the adult human brain will facilitate the understanding of GBM genesis and development of potential novel therapeutic targets.
STEM-24. SINGLE-CELL ATLAS OF HUMAN FETAL CEREBELLUM DECODING MEDULLOBLASTOMA ORIGIN AND ONCOGENESIS
Authors: Luo Zaili , Lu Richard
Published: November 2022

Medulloblastomas (MBs) are the most common malignant childhood brain tumors, yet the origin of the most aggressive subgroup-3 form remains elusive, impeding development of effective targeted treatment strategies. Previous cell-type analyses of mouse cerebella or human counterparts from frozen tissue nuclei have not fully defined the compositional heterogeneity of MBs. Here, we undertook an unprecedented single-cell profiling of freshly-isolated human fetal cerebella at different developmental stages to establish a reference map for delineating the hierarchical cellular states in MBs. We identified a unique transitional cerebellar progenitor connecting neural stem cells to neuronal lineages in human fetal cerebella. Intersectional analysis revealed that the transitional progenitors were enriched in aggressive MB subgroups, including group-3 and metastatic tumors. Integrated single-cell multi-omic profiling revealed unique regulatory networks in the transitional progenitor populations, including transcriptional determinants HNRNPH1 and SOX11, which are correlated with clinical prognosis in aggressive group-3 MBs. Genomic profiling and Hi-C analyses identified de novo long-range chromatin loops juxtaposing HNRNPH1/SOX11-targeted super-enhancers to cis-regulatory elements of MYC, an oncogenic driver for group-3 MBs. Targeting the transitional progenitor regulators inhibited MYC expression and MYC-driven group-3 MB growth. Together, our integrated single-cell atlases of human fetal cerebella and MBs reveal important cell populations predisposed to transformation and regulatory circuitries underlying tumor cell state evolution and oncogenesis, highlighting hitherto unrecognized transitional progenitor intermediates predictive of disease prognosis and potential therapeutic vulnerabilities.
STEM-28. THE ROLE OF LONP1 IN DRIVING ENHANCED PMT IN THE 'LEADING EDGE' NICHE IN GLIOBLASTOMA
Authors: Lepe Javier , Douglas Chris , Di Kaijun , Nandwana Nitesh , Das Bhaskar , Bota Daniela
Published: November 2022

Glioblastoma (GBM), a high grade brain tumor, possesses poor overall survival with less than 5% surviving past five years. Previously, the TCGA classifications for GBM have included the mesenchymal, proneural, classical and neural subtypes with their own respective expression profiles and survival. Recent omics analysis has revealed other key aspects of GBM pathology, including intratumoral heterogeneity spanning all subtypes and enhanced stemness and treatment resistance and other hallmarks of proneural mesenchymal transition (PMT) following treatment with first-line standard of care treatment with radiation therapy and temozolomide (TMZ). Invading glioma stem cells (GSC) with high Nestin and hypoxia-inducible factor 1 alpha (HIF-1α) expression have been theorized to contribute to recurrence. HIF-1α acts as a master regulator driving increased stemness, invasiveness and angiogenesis. Interestingly, HIF-1α and nuclear respiratory factor-2 both upregulate Lon peptidase 1 (LonP1) in response to increased hypoxia or reactive oxygen species (ROS) production. LonP1 has been shown to drive increased metastasis, tumor growth and epithelial-mesenchymal transition (EMT), an analog of PMT, in colon cancer, melanoma and other cancer types. In a recently elucidated GBM organoid model, we present new findings demonstrating the importance of LonP1 in driving enhanced, transient PMT near the ‘invading edge’. This includes the enhanced expression of several key drivers of PMT and phenotypic hallmarks, such as increased invasiveness, proliferation and poorer survival.
CSIG-01. EGFR AND AXL RECEPTOR TYROSINE KINASES DRIVE ONCOGENESIS BY LZTR1 MUTATION
Authors: Ko Aram , Hasanain Mohammad , Oh Young Taek , D'Angelo Fulvio , Sommer Danika , Frangaj Brulinda , Tran Suzanne , Bielle Franck , Pollo Bianca , Paterra Rosina , Mokhtari Karima , Soni Rajesh , Peyre Matthieu , Eoli Marica , Kalamarides Michel , Sanson Marc , Iavarone Antonio , Lasorella Anna
Published: November 2022

LZTR1, the substrate-specific adaptor of a CUL3-dependent ubiquitin ligase is among the most frequently mutated ubiquitin ligase coding gene in syndromic and sporadic human cancers including glioblastoma multiforme, in which approximately 27% of cases harbor inactivating mutations and copy number loss. However, both the identity of the protein substrates targeted by LZTR1-mediated ubiquitylation and the biological contexts regulated by specific LZTR1-substrate(s) interactions remain uncertain. Here, we combined biochemical and genetic studies to identify LZTR1 substrates and interrogated their tumor-driving function in the context of LZTR1 loss-of-function mutations and in a new conditional Lztr1 knockout mouse. Multiple screens converged on the receptor tyrosine kinases EGFR and AXL as LZTR1 interactors targeted for ubiquitin-dependent degradation in the lysosome by LZTR1-CUL3 complexes. Pathogenic mutations affecting LZTR1 failed to promote degradation of EGFR and AXL in human tumors. Mice harboring conditional deletion of the LZTR1 gene combined with loss of CDKN2A in the neural progenitor compartment generated peripheral nervous system tumors including schwannoma like and malignant peripheral nervous system tumors (MPNST). Tumors from the LZTR1-mutant mouse model accumulated very high levels of EGFR and AXL and exhibited potent and specific vulnerability to the combinatorial inhibition of EGFR and AXL kinases. These findings explain the mechanism of tumorigenesis associated with LZTR1 inactivation and offer a therapeutic strategy to patients affected by tumors carrying mutations of LZTR1.
CSIG-11. HYPOXIA SIGNALING IN ACVR1-MUTANT DIFFUSE INTRINSIC PONTINE GLIOMA
Authors: Johansson Elinn , Pietras Alexander
Published: November 2022

Diffuse intrinsic pontine glioma (DIPG) is a devastating glial tumor of the brainstem primary occurring in the pediatric population. The majority of DIPG tumors (60-80%) carry lysine- to methionine point mutation in histone H3, resulting in a global reduction of H3K27 trimethylation and subsequently a unique epigenetic landscape. Moreover, a large subset of the DIPG tumors carry activating mutations in ACVR1 – the gene encoding for the BMP receptor ALK2, suggesting an oncogenic role of signaling from this receptor. Importantly potential mechanisms of ACVR1-driven oncogenesis, remain poorly understood. Similar ACVR1 mutations have been described in the rare disorder Fibrodysplasia Ossificans Progressiva (FOP), and evidence from FOP models have suggested several links between hypoxia signaling, BMP signaling, and FOP pathogenesis thus opening up for evaluation of hypoxia signaling as oncogenic effector in ACVR1mutant DIPG. Using cultured DIPG cells and adult glioma cells respectively we have tested the activity of the hypoxia pathway using a hypoxia-responsive elements (HRE)-luciferase reporter assay. Intriguingly, DIPG but not glioma cells overexpressing a mutant ACVR1 construct significantly elevated hypoxia signaling at hypoxia. Similar trends are seen when assaying neuronal stem cell cultures harboring Histone 3.1 mutations along with activated ACVR1/ALK2 signaling. Furthermore, BMP ligand stimulation resulted in a dose-dependent increase of hypoxia signaling in DIPG cells. Through querying clinical gene expression data, we found elevated hypoxia signature scores in pediatric gliomas with mutant ACVR1 as compared to those with wildtype ACVR1. Finally, ACVR1 mutations frequently co-occur with Histone 3.3 and 3.1 mutations, and the hypoxia signature score was significantly higher than baseline only in tumors also harboring these Histone 3.3 or 3.1 mutations. Together, our data suggest that hypoxia signaling is elevated downstream of ACVR1 specifically in pediatric gliomas and prompts for evaluation of hypoxia signaling as potential therapeutical target in DIPG.
CSIG-25. DIFFERENTIAL YAP ACTIVITY IN HUMAN GLIOBLASTOMA TUMORSPHERES AS A POTENTIAL BIOMARKER
Authors: Kim Eui Hyun , Kang Seok-Gu
Published: November 2022

BACKGROUND

Hippo/YAP signaling pathway has emerged as an important driver of GBM. However, the clinical significance and expression correlation of YAP in GBM is still unknown. The purpose of this study is to elucidate the regulatory functions of YAP in response to tumorspheres (TS) in GBM. Material and

METHODS

In GBM-tumorspheres (TS), mRNA levels of Yap1 and TAZ were determined by RNA-Seq, and nuclear YAP1 expression level and its correlation with tumor aggressiveness were assessed through nucleus cytosolic fractionation, quantitative confocal microscopy, western blot, and TEAD4 reporter assay. Cell proliferation, stemness, and invasive properties were also measured using YAP siRNA and its inhibitor Verteporfin to reveal the effect of YAP on glioma progression. In orthotopic xenograft models, the inhibition of YAP function were tested.

RESULTS

All GBM-TS exhibited various phosphorylation states of YAP but no significant differences regarding the expression of YAP. Among them, TS15-88 cell line showed relatively low YAP phosphorylation in the quantified immunoblot results. Indeed, TS with reduced YAP phosphorylation was associated with aggressive cancer phenotypes. Predominant nuclear localization of YAP and binding of YAP/TAZ to TEAD in the nucleus was evident in the TS15-88 cells. The YAP knockdown (KD) by siRNA significantly resulted decrease in TS proliferation through WST and ATP assay and values were validated again through YAP inhibitor, Verteporfin (VP). YAP KD attenuated not only the invasion and stemness of TS but also caused a reduction in tumor volume and prolonged the overall survival of mice.

CONCLUSION

We showed alteration of gene activities by YAP and nuclear localization of YAP/TAZ complex contributes to GBM progression. Moreover, thus tuning of Yap activity results a marked suppression in aggressive phenotypes of cancer. These associations emphasize that the YAP signaling network highly suggests new therapeutic opportunities in diagnosing and treating GBM.
CSIG-29. EMPLOYING THE ZIKA VIRUS AS ONCOLYTIC VIROTHERAPY AGAINST PAEDIATRIC NERVOUS SYSTEM CANCER CELLS
Authors: Sherwood Matthew , Kaid Carolini , Mitsugi Thiago , Zhou Yilu , Wang Yihua , Skipp Paul , Gray Juliet , Okamoto Oswaldo , Ewing Rob
Published: November 2022

BACKGROUND

Malignant paediatric nervous system tumours, such as medulloblastoma, ATRT and high-risk neuroblastoma commonly harbour tumour cells with stem-like features which are highly tumorigenic and resistant to conventional therapies. These tumours can exhibit high lethality and may result in severe sequelae that significantly affect paediatric patients' quality of life. Oncolytic virotherapy exploits viruses that preferentially infect and destroy tumour cells. These viruses present a unique advantage in targeting highly heterogeneous cancers as they possess a secondary mechanism of action, through which they induce an anti-tumoral immune response. The Zika virus (ZIKV) is capable of infecting and destroying aggressive human paediatric brain tumour and neuroblastoma cells in vitro. ZIKV effectively reduces brain tumour size in mice (xenograft model) and canines (naturally occurring) and can induce an immune response against canine brain tumours.

METHODS

Employing global expression omics profiling of ZIKV infection and mapping of viral protein-host protein interactions, we aim to elucidate the mechanisms which underpin ZIKVs therapeutic properties, both at the molecular and cellular pathway levels.

RESULTS

Through extensive transcriptome profiling of ZIKV-infected paediatric brain tumour, neuroblastoma and NPCs, we have identified a variety of pathways which are involved in the ZIKV oncolytic response in the tumour cells and its neuro-dysregulation of NPCs. Despite both brain tumour and neuroblastoma cells undergoing ZIKV-induced oncolysis, we observed there to be a heterogeneous response within these different tumour cells at the molecular level to lead to oncolysis. Additionally, the infected tumour cells demonstrate elevated immune system profiles which alludes to the immune response that ZIKV may raise within the patient’s body against the paediatric tumour. Analysing our findings alongside the neuro-dysregulation we observe in our ZIKV-infected NPCs is allowing us to build a safety profile for employing a ZIKV-based therapy, whilst contributing to the growing knowledge of Congenital ZIKV Syndrome.
CSIG-37. MERLIN S13 DEPHOSPHORYLATION DRIVES MENINGIOMA WNT SIGNALLING AND CELL PROLIFERATION
Authors: Eaton Charlotte , John Liu S , Lucas Calixto-Hope , Casey-Clyde Tim , Choudhury Abrar , Daggubati Vikas , Vasudevan Harish , Swaney Danielle , Raleigh David
Published: November 2022

How Merlin-intact meningiomas arise in the absence of NF2/Merlin inactivation is incompletely understood. Here, we integrate single-cell RNA sequencing of 86,000 cells from meningioma xenografts with APEX2 proteomic proximity-labelling mass spectrometry and functional biochemical approaches to discover Merlin Serine 13 (S13) dephosphorylation drives meningioma Wnt signalling and cell proliferation. Cell biology, molecular biology, and biochemical techniques were used to validate Merlin functions in meningioma cells or xenografts using wildtype Merlin constructs or Merlin constructs encoding S13A, phosphomimetic S13D, or cancer-associated missense substitutions (L46R, A211D). Single-cell RNA sequencing of meningioma xenografts showed Merlin rescue activated the Wnt pathway in Merlin-deficient meningiomas. Proteomic proximity-labelling mass spectrometry revealed b-catenin, PKC, and PP1A interactions with wildtype Merlin, but not with Merlin L46R or A211D. b-catenin does not interact with other FERM family members, and Merlin contains a unique N-terminal domain (NTD) with a PKC phosphorylation motif overlapping with a PP1A dephosphorylation motif at S13. Thus, we hypothesized the Merlin S13, PKC, and PP1A may be important for Wnt signalling in Merlin-intact meningiomas. In support of this hypothesis, over-expression of wildtype Merlin or Merlin S13A but not Merlin DNTD, S13D, L46R, A211D, or other FERM family members drove meningioma Wnt signalling and sustained meningioma cell proliferation in vivo. Moreover, b-catenin was detected in proximity to Merlin S13D but not Merlin S13A in meningioma cells. Meningioma cell fractionation and immunofluorescence showed Merlin S13D over-expression stabilized b-catenin at the plasma membrane and inhibited Wnt signalling. Phospho-proteomic mass spectrometry and custom phospho-specific antibodies integrated with shRNA or siRNA gene suppression demonstrated PKC phosphorylated Merlin S13, but meningioma Wnt pathway activation induced PP1A to dephosphorylate Merlin S13 and drive cell proliferation. In summary, Merlin S13 dephosphorylation drives meningioma Wnt signalling and cell proliferation. These data reveal a novel tumor-promoting function of NF2/Merlin in Merlin-intact meningiomas.
CSIG-38. ENGINEERED PULLULAN-COLLAGEN-GOLD NANO COMPOSITE IMPROVES MESENCHYMAL STEM CELLS NEURAL DIFFERENTIATION AND INFLAMMATORY REGULATION
Authors: Chen Tracy , Yang Meng-Yin
Published: November 2022

Tissue repair engineering supported by nanoparticles and stem cells is an efficient strategy for promoting the healing potential of damaged tissues. Tissue repair engineering supported by nanoparticles and stem cells is an efficient strategy for promoting the healing potential of damaged tissues. In this study, we prepared various nanomaterials including pure Pul, pure Col, Pul–Col, Pul–Au, Pul–Col–Au, and Col–Au to investigate their physicochemical properties, biocompatibility, biological functions, differentiation capacities, and anti-inflammatory abilities through in vitro and in vivo assessments. The physicochemical properties were characterized by SEM, DLS assay, contact angle measurements, UV-Vis spectra, FTIR spectra, SERS, and XPS analysis. Pul–Col–Au composite enhanced cell viability, promoted anti-oxidative ability for MSCs and HSFs, inhibited monocyte and platelet activation, induced the lowest cell apoptosis, facilitated the MMP activities and improved endothelialization. In conclusion, Pul–Col–Au nanoparticles are promising material for tissue regeneration.
CSIG-40. STEAROYL-COA DESATURASE 1 (SCD1) IS REQUIRED FOR WNT SIGNALING TO INDUCE AN APOPTOSIS IN IDH MUTANT GLIOMA
Authors: Zhang Lumin , Yamasaki Tomohiro , Dowdy Tyrone , Lita Adrian , Gilbert Mark , Larion Mioara
Published: November 2022

BACKGROUND AND HYPOTHESES

SCD1, a major enzyme of saturated fatty acids, has been implicated to be important for tumor metabolic reprograming. Our previous study show that a high level of SCD1 mRNA is associated with IDH1mut lower grade gliomas. IDH1mut glioma cells are more sensitive to SFA induced apoptosis. However, the underlying mechanism remains unclear. In this study, we investigate the functions of SCD1 in IDHmut glioma and the potential contribution of SCD1 for cancer therapy of glioma.

STUDY DESIGN AND METHODS

The genetically engineered IDH wild-type, IDHmut and patient derived glioma cell lines were used to evaluate SCD1 functions. The expression of proteins were checked by Western-blotting assay. SCD1 was silenced by CRISPR or siRNA. The transcriptome change after SCD1 knockdown was profiled by RNA-seq or single cell RNA-seq (scRNA-seq).

RESULTS AND CONCLUSIONS

SCD1 transient silencing slowed down the cell growth, suggesting that SCD1 may possess an oncogenic property. RNA-seq analysis revealed that SCD1 inhibition decreased the expression of wnt-signaling pathway genes in IDH-1mut cells. scRNA confirmed that CRISPR SCD1 significantly decreased wnt signaling in the patient cell line Ts603. Therefore, we activated wnt pathway using a small chemical compound, BML-2838. Consistent with recent studies, wnt pathway induction led to a dramatically suppression of glioma cells growth. However, SCD1 silencing reversed this inhibitory effect. Further investigation revealed that SCD1 inhibition reduced the nucleus translocation of phosphorylated beta-catenin. Overall, the results suggest that SCD1 is vital for the onset of wnt pathway in glioma cells. High level of SCD1 expression may render the IDHmut glioma cells more sensitive to wnt pathway induced apoptosis.

RELEVANCE AND IMPORTANCE

In clinical, the 5-years survival rate of glioma remains low. SCD1 have been considered as a target for glioma therapy, recently. Our data provides a new insight on the strategy to target SCD1 in clinical.