bluuebunny/crossref_metadata_2025_split huggingface dataset:
MODL-23. PAIRED GLIOBLASTOMA CELL CULTURES OF THE FLUORESCENT BULK TUMOR AND NON-FLUORESCENT TUMOR MARGIN DISPLAY DIFFERENTIAL PHENOTYPES AND CELL STATES ACROSS PATIENTS
Authors: Neves Inês , Maturi Nagaprathyusha , Dang Yonglong , Lu Xi , Latini Francesco , Yildirim Irem , Sundström Anders , Jokinen Veera , Bergström Tobias , Xing Pengwei , Jarvius Malin , Larsson Rolf , Fryknäs Mårten , Ryttlefors Mats , Chen Xingqi , Swartling Fredrik , Uhrbom Lene 
Published: November 2023

Abstract
Glioblastoma is a devastating disease with a median survival of 8 months after diagnosis. Over 90% of patients have a recurrence that is in contact with the resection cavity. To understand the potential relapse-causing cells of the local infiltrative tumor margin, which have rarely been studied, we have established and analyzed paired cell cultures from the fluorescent bulk tumor and non-fluorescent tumor margin. Tumor samples were resected using 5-ALA fluorescence-guided surgery and employing stringent procedures to separate margin samples from bulk tumor cells. All margin samples were resected at least 1cm beyond the fluorescent border in non eloquent areas as determined intraoperatively. Bulk and margin samples from 13 glioblastoma patients were explanted and 7 paired cultures that could be maintained to at least passage 7 were established. Analysis of self-renewal and orthotopic tumor formation of both acutely dissociated cells and established cultures showed that bulk tumor cells in all cases had a higher capacity than their paired margin cells. Conversely, margin cells always displayed a higher invasive capacity than their paired bulk tumor cells. Combined single nucleus RNA- and ATAC-sequencing was performed on three paired cultures. Analysis of snRNA-seq data showed that the margin cell phenotype was connected with transitions towards more differentiated (AC- and MES-like) cell states compared to paired bulk tumor cells that showed a higher rate of cells in progenitor cell-like states (NPC- and OPC-like). snATAC analysis uncovered that the margin cell phenotype was connected with transcription factor (TF) motifs known to be involved in cell invasion and differentiation, while paired bulk tumor cells were enriched for TF motifs involved in neural development and proliferation. We present a unique collection of margin cell cultures that we believe are more relevant models of the relapse-causing glioblastoma cells than those established from the bulk tumor.
MODL-25. INTERNAL CAROTID INJECTION MODEL OF BRAIN METASTASIS DESCRIBES LEPTOMENINGEAL DISEASE
Authors: Chernikova Sophia , Tsau Sheila , Wang Yuelong , Hartmann Griffin , Polyak Dina , Bhambhvani Hriday , Kolluru Sai Saroja , Trinh Thy , Casey Kerriann , Johnson Eli , Connolly Ian , Recht Lawrence , Ahn G-One , Nagpal Seema , Quake Stephen , Brown J Martin , Gephart Melanie Hayden 
Published: November 2023

Abstract
Leptomeningeal disease (LMD), the most aggressive form of central nervous system (CNS) metastasis in which cancer cells infiltrate leptomeninges, is universally and rapidly fatal due to poor detection and no effective treatment. Treatment of LMD is challenged by the diffuse nature of the disease, low specificity of detection, poor drug penetration into CNS and high neurotoxicity. LMD is a heterogeneous disease: co-existence of leptomeningeal spread and parenchymal brain metastases in not uncommon, and LMD tumors vary in size and may be found at various locations throughout entire neuroaxis. Radiographic detection (i.e., MRI) works well for big “bulky” metastases but may produce normal results in patients with active disease identified by cerebrospinal fluid (CSF) tap. This spatial and morphological heterogeneity of LMD may ultimately be responsible for a varied and limited therapy response. Mechanistic and translational studies of LMD are hampered by the lack of adequate in vivo models that recapitulate the consecutive steps of LMD progression separate from systemic disease. In this study, we show that the internal carotid (ICA) injection of tumor cells, which is widely used to model brain metastasis, produces leptomeningeal spread. The ICA injection of various tumor cell lines faithfully recapitulated the stages, heterogeneity, and spatial distribution of human LMD. While some cell lines demonstrated clear preference for growth in brain parenchyma, they still had a measurable population of cells within the leptomeninges. Most bulky LMD metastases formed and grew near fenestrated endothelium, where the direct access to blood might support tumor growth and survival. A population not associated with the fenestrated endothelium consisted mostly of single non-dividing non-hypoxic cells. This diffuse cancer spread limited the efficacy of standard whole-brain radiation therapy. Thus, leptomeningeal spread should be taken into account when interpreting the data from the ICA model that is widely used to study brain metastasis.
MODL-26. A PROTOCOL TO ADAPT GLIOBLASTOMA IN VITRO CULTURE SYSTEMS INTO ORGANOID MODELS USING NEUROCULT™ NS-A PROLIFERATION MEDIUM
Authors: McCormack Kasandra , Mak Carmen , Eaves Allen , Louis Sharon , Knock Erin 
Published: November 2023

Abstract
Glioblastoma (GBM) is a malignant and aggressive brain tumor that is difficult to treat due to its heterogeneity. Traditional in vitro models (i.e. monolayers and tumorspheres) do not recapitulate this heterogeneity, leading to difficulty assessing new therapeutic strategies. Culturing GBM cells as organoids may provide a better method to retain the phenotype of the parent tumor. Here, we present a protocol for generating GBM organoids using NeuroCult™ NS-A Proliferation Medium, a medium commonly used for tumorsphere cultures. Two methods were assessed for the initial stage of formation, each suitable depending on throughput and cell source. In the first method, tumorspheres were dissociated and single cells were mixed with Growth Factor Reduced Matrigel® at a 1:4 ratio to form 15 μL droplets with 1000 cells per droplet. In the second method, 9000 cells from dissociated tumorspheres were seeded per well in a 96-well U-bottom plate and cultured for 5 days to form aggregates. On day 6, the aggregates were embedded in 15 μL Matrigel® droplets. In both methods, polymerized droplets were then cultured in suspension in stationary 6-well plates for 3 days, then maintained on an orbital shaker with full-medium changes every 2 - 3 days. Organoids were cryosectioned and processed for immunofluorescence at 5 - 6 weeks. In vivo, GBM tumors display an architecture containing regions of high and low nutrient and oxygen availability around a necrotic core. GBM organoids generated using this protocol exhibited distinct areas of proliferation near the organoid rim (Ki67+) and hypoxia (CA9+) near the core, mimicking this in vivo architecture (n = 3 GBM cell lines). Organoids grew to 2 - 4 mm in diameter and expressed glioma markers including TUJ1, Nestin, SOX2, OLIG2, and GFAP. These data show that NeuroCult™ NS-A Proliferation Medium can be used to adapt traditional GBM culture systems into organoids.
MODL-27. INTEGRATING OVERALL SURVIVAL AND TUMOR CONTROL PROBABILITY MODELS TO PREDICT LOCAL PROGRESSION AFTER BRAIN METASTASIS RADIOSURGERY
Authors: Quezada Jeffrey , Harris Jeremy , Healy Erin , Seyedin Steven , Chen Allen , Kuo Jeffrey , Simon Aaron 
Published: November 2023

Abstract

PURPOSE
Stereotactic radiosurgery (SRS) for brain metastases has competing goals of maximizing local control and minimizing toxicity. Tumor control probability (TCP) models predict decreasing risk of local progression (LP) with dose escalation. However, many patients die from extracranial disease prior to local progression and may not require maximally aggressive treatment, which increases risk of toxicity. Better tools to assess patients’ risk of LP after SRS could help practitioners personalize SRS treatment to maximize therapeutic ratio. Here we assess whether simultaneous modeling of TCP and overall survival (OS) can improve prediction of LP after SRS.

METHODS
Records of patients undergoing SRS at a single institution were reviewed retrospectively. Using established OS and TCP models, the probability of one-year survival [p(OS)] and probability of one-year LP [p(LP)]) were calculated for each patient and each metastasis, respectively. Joint-probability was used to combine the models [p(LP,OS)=p(LP)*p(OS)]. Analyses were conducted at the individual-metastasis and whole-patient levels. Fine-Gray regression was used to model the effect of p(LP) or p(LP, OS) on risk of local progression after SRS, with death as a competing risk.

RESULTS
Ninety-two patients and 391 lesions were identified. At the patient level, one-year LP was 0.08 (95%CI, 0.03-0.15), median p(LP,OS) was 0.13 (95%, CI 0.07-0.2), and median p(LP) was 0.29 (95% CI, 0.22-0.38). At the metastasis level, one-year LP was 0.02 (95%CI, 0.01-0.04), median p(LP,OS) was 0.05 (95%, CI 0.02-0.07), and median p(LP) was 0.10 (95% CI, 0.07-0.13). p(LP,OS) was significantly associated with the risk of local progression at the patient level (p=0.048) and metastasis level (p=0.007); however, p(LP) was not (p=0.16 and p=0.28, respectively).

CONCLUSION
This study suggests simultaneous OS and TCP modeling more accurately predicts LP than TCP modeling alone. Better understanding which patients are at risk of LP after SRS may help personalize treatment to minimize risk without sacrificing efficacy.
MODL-29. EXPLORING CELL STATE TRANSITIONS IN GLIOBLASTOMA WITH LIVE REPORTING OF CELL STATES USING CRISPR/CAS9
Authors: Stockgard Rebecka , Yildirim Irem , Kleindienst Sean , Heinold Josephine , Nelander Sven 
Published: November 2023

Abstract
Glioblastoma (GBM) is the most common malignant primary brain tumor, with a median survival time of 12-15 months. The heterogeneity of GBM, ranging from inter-patient to intra-tumoral, poses a significant challenge in identifying effective treatments. At the single-cell level, GBMs comprise a multiplicity of transcriptional cell states that can change over time and in response to therapy. To explore how cell state plasticity shapes tumor progression, new experimental tools to monitor cell state dynamics in real-time will be important, if not essential. Here, we describe a live reporting of GBM cell states that utilize CRISPR/Cas9 and fluorescent proteins for tracking individual glioblastoma patient-derived cells’ (GBM-PDC) physiological gene expression, and cell state transitions over time and during treatment. The method aims to provide real-time reporting of accurate transcriptional phenotypes (e.g. OPC/NPC-like, Mesenchymal, Astrocyte states) over extended periods or in relevant in vitro or in vivo contexts. Our protocols achieved successful endogenous tagging of a common stem cell marker with full-length mCherry in up to 15 % of GBM-PDC (an order of magnitude more effective than previous reports). Real-time imaging (Incucyte S3 and IMX-C) allows us to trace individual cells’ states over time and during treatment, supported by ICC, qPCR, and Western blot, providing a reliable approach. Our next objective is to employ this method to tag several cell-state marker genes with fluorescent proteins. We can directly follow gene expression and cellular state transitions when several state-selective genes are fused with different fluorescent proteins. We anticipate establishing a robust framework to investigate the temporal plasticity and invasion patterns within GBM cells across different patients which may open for new therapeutic opportunities.
MODL-31. USING GENETICALLY-ENGINEERED MOUSE MODELS OF TREATMENT-NAÏVE AND -EXPERIENCED NTRK FUSION-DRIVEN GLIOMAS FOR PRE-CLINICAL TRIALS OF TYROSINE KINASE INHIBITORS
Authors: Schmid Sebastian , Russell Zachary R , West Madeline , Walker Julia , Holland Eric , Szulzewsky Frank 
Published: November 2023

Abstract
Gene fusions that contain the kinase domain of the three neurotrophic tyrosine kinase receptor (NTRK) genes are frequently observed in infantile high and low-grade gliomas. While these tumors initially respond to treatment with NTRK tyrosine kinase inhibitors (TKIs), treatment resistance and recurrence eventually occur. Treatment-resistant tumors frequently harbor either on-target (in the fusion's kinase domain) or off-target mutations (in downstream effectors of mitogen-activated signaling pathways). Therefore, overcoming this resistance requires a three-pronged strategy: 1) identifying new kinase inhibitors capable of targeting NTRK fusions with on-target kinase domain mutations, 2) identifying druggable targets in the downstream pathways activated by these fusions, and 3) targeting persister cells that contribute to disease recurrence. We utilized the RCAS/tv-a system to establish several mouse models of NTRK1/2/3 fusion-driven gliomas. We found that all analyzed fusions were strong oncogenic drivers on their own, while the loss of additional tumor suppressors (Cdkn2a or Pten) further increased the aggressiveness of these tumors. These mouse models exhibit similar treatment responses as human tumors, initially responding to various TKIs but eventually recurring due to the presence of treatment-resistant persister cells. To mimic the emergence of resistance-associated mutations, we directly introduced different kinase domain mutations into the NTRK fusion sequence or co-expressed secondary mutations (BRAF-V600E or KRAS-G12D). These modifications altered the response to TKI treatment both in vitro and in vivo. Overall, these models serve as valuable tools for studying the biology of NTRK fusion tumors, their treatment response in a treatment-naïve or -experienced state, and the mechanisms underlying treatment resistance.
MODL-32. EGFRVIII OVEREXPRESSION AND LOSS OF MOUSE SPECIFIC CDKN2A IN GLIAL CELLS LEADS TO GLIOMAGENESIS IN A NOVEL MOUSE MODEL
Authors: Singh Olivia , Li Mira , Ali Hafsah , Voisin Mathew , Wei Qingxia , Gao Andrew , Agnihotri Sameer , Burrell Kelly , Wilson Mark , Kamiri Shirin , Aldape Kenneth , Zadeh Gelareh 
Published: November 2023

Abstract
A new mouse model for the classical subtype of human glioblastoma has been generated using Cre-mediated EGFRvIII overexpression and homozygous p19-ARF deletion (the mouse homolog of human p14-ARF/CDKN2A) in GFAP expressing cells. Transgenic mice develop intraparchenymal and/or leptomeningeal brain lesions with some spinal cord invasion as early as 1 month old and 95% of mice die by 6 months due to hydrocephalus and/or paralysis. Mice with high grade tumors have worse survival and similar features to human classical glioblastoma such as necrosis, high levels of mitosis, and infiltration of tumor cells into normal brain. Immunohistochemical analysis confirms EGFRvIII overexpression and p19-ARF loss in tumor cells, along with patchy positive GFAP and positivity for Olig2, S100β and NeuN, suggesting that tumor cells arise from a progenitor glial cell type. Adherent and neurosphere primary culture of dissociated tumors indicate that tumor cells maintain EGFRvIII expression in culture and are able to generate xenograft tumors by 3 weeks after intracranial injections into NODSCID mice. Xenograft tumors are reminiscent of the primary tumor, with similar histopathological features and immunohistochemical staining. This novel mouse model can be used to study diffuse glioma with a leptomeningeal component.
MODL-33. PATIENT-DERIVED GLIOBLASTOMA MINIATURE AND CANCER STEM CELL SUICIDE-INDUCING NANO-AWAKENER BASED ON IT
Authors: Yoon Seon-Jin , Baek Sewoom , Yu Seung-Eun , Jo Euna , Choi Ran Joo , Moon Ju Hyung , Kim Eui-Hyun , Chang Jong Hee , Sung Hak-Joon , Kang Seok-Gu 
Published: November 2023

Abstract
Patient-specific anti-tumor therapies can evolve by vitalizing the mother tissue-like cancer niche, cellular profile, genetic signature, and drug responsiveness. This evolution elucidated a key mechanism along with the development of the cancer therapy. The present study aimed to meet these clinical demands by approaching the direct culture of GBM patient tissues with a 1 mm post-punching diameter in a 3D microchannel network chip (“GBM miniature”). Hence, we directly cultured of GBM patient tissue (1 mm diameter) in a microchannel network chip (GBM miniature) to preserve mother tissue-like characteristic signatures and microenvironment. When chemoradiation therapy were administered within 1 day, the responsiveness of the tissue in the chip mirroring the clinical outcomes. The characteristics of migrating cells from GBM miniature were analyzed, and peptides that target these migrating CSCs were searched for. And, by attaching this CSC peptide to the nanovesicle, the therapeutic effect was confirmed in GBM miniature. Upon testing within 1 day, the tissue chip reflected patient drug responsiveness as seen in the clinic. As the tissue chip culture was continued, the intact GBM signature was getting lost, and the outward migration of stem cells from the tissue origin increased, indicating a leaving-home effect on the family dismantle. Nanovesicle production using GBM stem cells enabled self-chasing of the cells that escaped the temozolomide effect owing to quiescence. The anti-PTPRZ1 peptide display and temozolomide loading to nanovesicles awaked stem cells from the quiescent stage to death. This research proposes a GBM patient avatar platform and mechanism-learned nanotherapy for translation.
MODL-38. DEFINING THE ROLE OF DAAM2 R414W MUTATION IN GLIOMAGENESIS AND GBM GROWTH
Authors: Choy Tiffany , Ye Qi , Deneen Benjamin , Bondy Melissa , Lee Hyun Kyoung 
Published: November 2023

Abstract
Each year in the United States, more than 13,000 people are diagnosed with glioblastoma multiforme (GBM), and more than 10,000 are estimated to succumb to the disease, making GBM the most common and lethal primary adult brain tumor. Despite advancing therapies, GBM patient survival has not improved over the decades, so it is critical to better understand glioma etiology. The majority of GBM cases arise from a sporadic accumulation of somatic mutations, but approximately 5-10% of gliomas are classified as “familial”, occurring clustered with hereditary cancer predispositions. These familial gliomas are caused by germline mutations inherited from parent to offspring, and only a few of these mutations have been directly linked to the development of GBM. We recently discovered five germline mutations in Daam2 (Dishevelled associated activator of morphogenesis 2) from GBM patients with a history of familial glioma. To examine the role of these mutations in GBM, we overexpressed these mutations in patient-derived GBM cells, which were then orthotopically xenografted into immunodeficient mice. We identified that one of these mutations, Daam2-R414W, promotes tumor cell proliferation both in vitro and in vivo. To further understand the role of the Daam2-R414W mutation in GBM, we generated a knock-in immunocompetent mouse model that harbors the Daam2-R414W mutation. In these mutant mice, we found increased proliferation of glial progenitor cells in the subventricular zone compared to Daam2 wildtype mice, suggesting the potential of the Daam2-R414W mutation to influence glial progenitor expansion to stimulate glioma initiation. Future experiments aim to answer whether this mutation promotes GBM growth in an immunocompetent model and to decipher the mechanism relating the Daam2-R414W mutation to GBM pathology. These studies will shed light on the repercussions of a human developmental gene variant and its potential to disrupt normal cellular machinery to promote malignancy and GBM development.
MODL-41. LOSS OF DISTAL CHROMOSOME 6Q AND ASSOCIATED PDE10A INDUCES AN AGGRESSIVE GLIOMA PHENOTYPE BY FACILITATING PRONEURAL TO MESENCHYMAL TRANSITION
Authors: Nuechterlein Nicholas , Shelbourn Allison , Alvinez Neriah , Szulzewsky Frank , Arora Sonali , Paddison Patrick , Merino-Galan Leyre , Pattwell Siobhan , Sulman Erik , Jung Mi-Yeon , Brown Desmond , Banasavadi-Siddegowda Yeshavanth Kumar , Tang Kayen , Jackson Sadhana , Stoica Stefan , Chittaboina Prashant , Patel Anoop , Holland Eric , Cimino Patrick , Shapiro Linda 
Published: November 2023

Abstract
Somatic copy number alterations (SCNAs) are common in glioblastoma, with some SCNAs having prognostic implications. Identifying and characterizing SCNA-related genes acting as oncogenes or tumor suppressors may potentially provide new therapeutic avenues for gliomas. In this study, we sought to uncover novel SCNA-related genes by employing an unbiased log rank analysis screen of genomic regions associated with glioblastoma survival. We identified 6q27 loss as an independent poorly-prognostic SCNA across three separate glioblastoma cohorts. An in vitro CRISPR screen using human neural stem cells identified five genes in the 6q27 region that caused increased cellular proliferation. One of these genes, phosphodiesterase 10A (PDE10A), had a gene dosage effect on expression and decreased expression in human and mouse glioma tumor tissue compared to normal brain, which are both common tumor suppressor phenotypes. Knockdown of Pde10a in mouse neural stem cells in vitro validated the human cell CRISPR screen phenotype. Suppression of Pde10a in RCAS/Tva mouse glioma in vivo caused decreased survival with increased tumor grade, mitotic activity, necrosis, Vegfa expression, and AKT/pS6 signaling. Single cell RNA-sequencing of mouse gliomas revealed that Pde10a suppression results in a shift from proneural to mesenchymal-like state. In human glioma tissue, PDE10A loss is also associated with an increased mesenchymal signature. Top activated pathways in Pde10a suppressed gliomas involved cell-cell interaction, including upregulation of cell adhesion molecules and downregulation of focal adhesion complex disassembly genes. Functional analysis in vitro confirmed that Pde10a suppression results in increased extracellular matrix adhesion and decreased migration. Relevant to standard glioblastoma treatment, PDE10A loss was associated with decreased MGMT promoter methylation, decreased DNA methyltransferase gene expression, and it potentiated radiation resistance in vitro. In summary, we leveraged several multiomic and experimental platforms to reveal PDE10A as a tumor suppressor in glioblastoma, predominantly by inducing a proneural to mesenchymal transition phenotype.