Human Glioblastoma Research Articles

Synthesis and effect of 4-acetylphenylamine-based imidazole derivatives on migration and growth of 3D cultures of breast, prostate and brain cancer cells.

In this study, we have synthesized novel 4-acetophenone moiety-bearing functionalized imidazole derivatives containing S-, and N-ethyl substituents and evaluated their anticancer activity. Their anticancer activity was studied against human breast carcinoma (MDA-MB-231), human prostate carcinoma (PPC-1), and human glioblastoma (U-87). Compounds 4, 9, 14, and 22 were identified as the most promising anticancer agents from a series of imidazole derivatives. They showed the highest cytotoxicity by MTT assay against MDA-MB-231, PPC-1 and U-87 cell lines. Compounds 14 and 22 were most selective against PPC-1 and U-87 cell lines, and their EC50 values against these cell lines ranged from 3.1 to 47.2 µM. Most tested compounds showed lower activity against the triple-negative breast cancer MDA-MB-231 cell line. None of the imidazole derivatives possessed an inhibiting effect on the migration of PPC-1 and U-87 cells by 'wound' healing assay. In spheroid assay, the most promising were compounds 14 and 22, especially in PPC-1 3D cultures. They efficiently reduced both the size and the viability of PPC-1 spheroid cells.

Adenoviral delivery of the CIITA transgene induces T-cell-mediated killing in glioblastoma organoids.

The immunosuppressive nature of the tumor microenvironment poses a significant challenge to effective immunotherapies against glioblastoma (GB). Boosting the immune response is critical for successful therapy. Here, we adopted a cancer gene therapy approach to induce T-cell-mediated killing of the tumor through increased activation of the immune system. Patient-based three-dimensional (3D) GB models were infected with a replication-deficient adenovirus (AdV) armed with the class II major histocompatibility complex (MHC-II) transactivator (CIITA) gene (Ad-CIITA). Successful induction of surface MHC-II was achieved in infected GB cell lines and primary human GB organoids. Infection with an AdV carrying a mutant form of CIITA with a single amino acid substitution resulted in cytoplasmic accumulation of CIITA without subsequent MHC-II expression. Co-culture of infected tumor cells with either peripheral blood mononuclear cells (PBMCs) or isolated T-cells led to dramatic breakdown of GB organoids. Intriguingly, both wild-type and mutant Ad-CIITA, but not unarmed AdV, triggered immune-mediated tumor cell death in the co-culture system, suggesting an at least partially MHC-II-independent process. We further show that the observed cancer cell killing requires the presence of either CD8+ or CD4+ T-cells and direct contact between GB and immune cells. We did not, however, detect evidence of activation of canonical T-cell-mediated cell death pathways. Although the precise mechanism remains to be determined, these findings highlight the potential of AdV-mediated CIITA delivery to enhance T-cell-mediated immunity against GB.

TMOD-31. SINGLE-CELL PROFILING AND CHARACTERIZATION OF PATIENT-DERIVED XENOGRAFT GLIOBLASTOMA MODEL IN HUMANIZED MICE

Abstract Glioblastoma (GBM) is the most common and aggressive malignant brain tumor in adults, and the current standard therapy has limited efficacy. To develop effective therapies, it is desirable to develop an animal model that can replicate the tumor heterogeneity and immune microenvironment of human GBM. Here, we describe a novel GBM mouse model established with a patient-derived xenograft (PDX) in humanized mice harboring a nearly complete human immune environment. Humanized mice were generated by engrafting human CD34+ hematopoietic stem cells from umbilical cord blood into immunocompromised mice (NSG, NSG-SGM3, and NOG-EXL) after myeloablation. NSG-SGM3 is a triple transgenic mouse encoding human IL3, GM-CSF, and SCF on the NSG strain background. NOG-EXL is a NOG strain with human IL3 and GM-CSF expression. These cytokines foster the development of the human myeloid lineage and its differentiation. NSG-SGM3 and NOG-EXL were superior in immune cell reproducibility, especially myeloid cells. Radioresistant PDX GBM cells established after repeated irradiation were injected into the forebrain of humanized mice and the same immunocompromised mice without humanization. Human PDX GBM cells formed aggressive tumors with similar growth speed in the humanized mice compared to non-humanized mice; however, their dissemination to the spinal cord was significantly suppressed in humanized mice, suggesting that human immune cells impact the spinal dissemination of GBM cells. Single-cell RNA sequencing analysis revealed infiltration of human CD4+ T, CD8+ T, NK cells, and macrophages, as well as infiltration of regulatory T and exhausted CD8+ cells into the brain tumor, reflecting the immunosuppressive landscape of recurrent human GBM. Furthermore, we found that tumor heterogeneity was increased in humanized mice compared to non-humanized mice. There was a notable rise in neural progenitor-like cell populations, indicating tumor adaptation to immune pressures. This novel GBM-PDX humanized mouse model might be highly beneficial for analyzing the interaction between human tumor cells and human immune cells, and evaluating the effect of immunotherapies.

TMOD-35. EGFRVIII OVEREXPRESSION AND LOSS OF MOUSE SPECIFIC CDKN2A IN GLIAL CELLS LEADS TO SPONTANEOUS GLIOMAGENESIS IN A NOVEL MOUSE MODEL

Abstract A new mouse model for the classical subtype of human glioblastoma (GBM) has been bred using Cre-mediated EGFRvIII overexpression and homozygous p19-ARF deletion, the mouse homolog of human p14ARF/CDKN2A, in GFAP expressing cells. Transgenic mice develop intraparenchymal 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 GBM 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 Cre-expressing cells, along with patchy positive GFAP and high proliferation by Ki67. Bulk RNA sequencing reveals GEC3 tumors cluster closely with human classical GBM and have significant upregulation of downstream markers in the JAK/STAT and P13K/AKT pathways. Adherent and neurosphere primary culture of dissociated tumors indicate that tumor cells maintain EGFRvIII expression in culture and 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. Our spontaneous glioma model is a powerful tool for future studies focused on the role of the immune microenvironment on glioma recurrence and resistance.

EXTH-65. EXPLORING ANTIHISTAMINES TO TREAT GLIOBLASTOMA

Abstract BACKGROUND Glioblastoma (GBM) is an aggressive and lethal primary malignant brain tumor with a 5-year survival of 6.8%. Interestingly, people with allergies, atopy, or asthma are less likely to develop GBM. A recent study recognized that high histamine receptor 1 (HRH1) expression is inversely related to overall and progression-free survival. METHODS We used a bioinformatics tool, iLINCS, which, after analyzing 99 GBM and 38 healthy samples, identified 36 drugs that can reverse GBM signatures. Based on the concordance score and blood-brain barrier permeability, we selected brompheniramine (Brom), a first-generation HRH1 antagonist, as a potential candidate. HRH1 expression analysis was done on human and mouse GBM cell lines by immunoblotting. The effect of Brom alone or in combination with temozolomide (TMZ) was investigated on proliferation, DNA damage and apoptosis in human (U118 and U251) and three syngeneic (EGFRvIII, p16-/- & GFAP Cre, PTEN+/-, p53R172H+/-, EGFRvIII & GFAP Cre, PTEN-/-, p53R172H-/- & GFAP Cre) cell lines. GBM orthograft mouse models were used to test the efficacy of combination therapy. RESULTS HRH1 is highly expressed in human and syngeneic GBM cells. This expression is further increased in TMZ-resistant cells. Interestingly, the addition of histamine, induced TMZ resistance in GBM cells. TMZ with Brom decreases proliferation in mouse and human GBM cells, and Brom is non-toxic to non-transformed cells. Combination treatment increases DNA damage and decreases the DNA repair mechanism, possibly by downregulating the expression of O6methylguanine-DNA methyltransferase (MGMT). In the GBM orthograft mouse model, the combination therapy reduced tumor volume and significantly increased survival compared to single-agent treatment. CONCLUSIONS Brom is non-toxic; Brom, in combination with TMZ, enhanced the anti-tumor efficacy of TMZ both in in vitro and in vivo GBM models. Further studies in spontaneous mouse models are planned.

STEM-04. UNVEILING GLIOBLASTOMA ORIGIN-CELLS: ISOLATION AND CHARACTERIZATION FROM THE SUBVENTRICULAR ZONE

Abstract Glioblastoma (GBM) may originate from somatic mutations within the subventricular zone (SVZ). We investigated putative GBM origin-cells in the SVZ with somatic mutations implicated in GBM development. We hypothesized these origin-cells have unique properties compared to tumor-derived GBM tumorspheres (TSs). We isolated, characterized, and analyzed mouse transcriptomes of GBM origin-cells and GBM TSs, and sought to identify potential human GBM origin-cells within the SVZ. Using a mouse model with somatic mutations (Trp53/Pten/hEGFRvIII) induced within the SVZ, we demonstrated that mutated cells in the SVZ migrate to the cortex and form GBM. We isolated mouse GBM (mGBM) origin-cells from the SVZ and mGBM TSs from the tumors, comparing their characteristics, transcriptome, and tumorigenic potential upon orthotopic or SVZ injection. To identify potential human GBM origin-cells, single-nucleus RNA sequencing (snRNAseq) was performed on human GBM SVZ specimens. Our investigation revealed intriguing properties and distinct gene expression patterns in GBM origin-cells. mGBM origin-cells from the SVZ somatic mutation model demonstrated enhanced stemness and invasiveness compared to control SVZ cells. These cells exhibited site-specific tumorigenic capacity: cortical injection of mGBM origin-cells failed to induce tumor formation, while re-injection into the SVZ led to migration to the cortex and tumor development. RNA sequencing revealed significant gene expression differences between mGBM origin-cells and mGBM TSs despite similar mutation profiles, suggesting distinct identities. snRNAseq analysis of human GBM SVZ specimens (pathologically tumor-free) identified cells with chromosomal aberrations characteristic of early GBM development, including chromosome 7 gain / 10 loss. These findings suggest potential human GBM origin-cells within the SVZ. This study describes a distinct population of GBM origin-cells within the SVZ, different from tumor-derived cancer stem cells. These GBM origin-cells exhibit unique properties, including enhanced stemness and invasiveness, and display site-specific tumorigenic capacity, forming tumors only in the SVZ environment. Further research into the factors that cause GBM origin-cells in the SVZ to migrate and form tumors in the cortex is crucial.

TMET-32. THE ROLE OF HOXC10 IN DRIVING GLIOBLASTOMA AGGRESSIVENESS

Abstract BACKGROUND Glioblastoma (GBM) is an aggressive malignancy, with a tumor doubling time of just 49.6 days. Despite maximal safe surgical resection, radiation, and temozolomide, median survival is only 14.6 months. Homeobox gene, homeobox C10 (HOXC10), a transcription factor of the HOX10 family, is pivotal in neuronal development, upregulated in several cancers, including glioma, and correlates with poor survival. METHODS RNA sequencing analysis was done on our mouse glioma syngeneic cell lines. HOXC10 expression analysis was done on human and mouse GBM cell lines and tissues by immunoblotting and immunohistochemistry, respectively. HOXC10 and p16-/-silencing were done using 4 unique 29mer shRNA constructs in the GFP vector, respectively. Gene Ontology predicted HOXC10 functions. HOXC10 and pyrroline-5-carboxylic acid reductase 1 (PYCR 1), a proline biosynthetic enzyme co-expression was studied using immunofluorescence analysis. RESULTS HOXC10 is highly expressed in human and mouse GBM tissues. HOXC10 was upregulated only in the high-grade/aggressive cell line [EGFRvIII; p16-/- & GFAP Cre] and demonstrated increased expression with p16 deletion in the mouse glioma cell line. Compellingly, TCGA analysis of GBM correlated the negatively prognostic p16 homozygous deletion (HD) with HOXC10 upregulation. Gene Ontology supports the role of HOXC10 for proline biosynthesis and metabolism. Immunofluorescence on mouse GBM sections revealed co-expression of HOXC10 and PYCR1. HOXC10 silencing significantly decreased GBM cell line proliferation, induced apoptosis, and decreased PYCR1 expression in vitro. CONCLUSIONS HOXC10 expression is increased in GBM and with p16 HD. HOXC10 silencing significantly decreased GBM cell line proliferation and decreased PYCR1 expression. HOXC10-mediated proline biosynthesis may present a novel target to improve GBM survival.

EPCO-47. SEX-BIASED BRD4 ACTIVITY DRIVES TUMORIGENESIS IN GLIOBLASTOMA

Abstract Consistent sex differences in incidence and outcome have been reported in cancers including brain tumors. Glioblastoma (GBM), the most common and aggressive primary brain tumor, occurs with higher incidence and shorter survival in males compared to females. Brd4 is essential for regulating transcriptome-wide gene expression and specifying cell identity. Using an isogenic murine GBM model, we have reported correlated transcriptome-wide sex differences in gene expression, Brd4-bound enhancer usage, and Brd4 localization to oncogenic and tumor suppressor transcription factors binding sites in male and female GBM cells, respectively. Sex-biased gene expression patterns were also evident in human glioblastoma. Inhibiting Brd4 function abrogates sex differences in these transcriptional states and concomitant differences in tumorigenic phenotype. Thus, sex-biased Brd4 activity drives sex differences in GBM rendering males and females differentially sensitive to BET inhibitors. These data indicate that Brd4 can exhibit dual functions as an oncogene or tumor suppressor, dependent upon cellular sex. To better understand how to globally target Brd4, we interrogated the role played by master transcriptional Brd4 regulators in coordinating this dual phenotype in GBM. Motif-based analysis revealed that Brd4 co-localized with oncogenes such as Bach1, and Klf5 at male-biased enhancers whereas female-biased enhancers were co-occupied with tumor suppressors, such as Smad4 and p53. Knockdown of Bach1 decreased clonogenic frequency in males and females. Conversely, knockdown of Smad4 increased proliferation and clonogenicity in males, further inducing the male tumorigenic phenotype, while no change was observed in females. These results indicate that Smad4 might be necessary but not sufficient to provide females with resistance to transformation. Ongoing epitranscriptomics investigating Brd4 re-localization and enhancer usage in knockdown cells will increase our understanding of Brd4 mechanism in GBM. Targeting sex-biased Brd4 function will identify novel combinatorial sex-specific therapeutics for brain tumors and could have immediate impact on the clinical treatment of GBM patients.

EXTH-15. AN EGFRVIII RNA-LNP VACCINE PROTOTYPE ACHIEVES TUMOUR CLEARANCE IN A GBM MOUSE MODEL

Abstract Glioblastoma (GBM) is an aggressive high-grade glioma that is fatal despite standard-of-care therapy. Recurrent somatic alterations that form neoantigens represent opportunities for targeted therapeutic intervention. Using human EGFRvIII as a prototypical structural alteration and neoantigen, we tested the potential of a mRNA-LNP vaccine approach to improve the survival of mice with EGFRvIII-driven GBM. To generate the model, we used in vivo electroporation to introduce genetic alterations (EGFRvIII-OE/CDKN2A-KO/PTEN-KO) into periventricular cells of early postnatal C3H mice, then propagated cells from the subsequent tumours to create implantable cell lines. Following orthotopic implantation, the resulting syngeneic tumours faithfully recapitulate histologic features of GBM with infiltrative growth, mitoses, necrosis, and vascular proliferation. Also like human GBM, the models are sensitive to but not cured by temozolomide chemotherapy and are resistant to immune checkpoint blockade. We delivered mRNA-LNP vaccines against EGFRvIII into the thigh muscle of tumour-bearing mice on days 7, 10, 14, 21, and 36 after tumour cell implant. We observed no tumour in vaccine-treated mice compared to significant tumour burden in control vector-treated animals by MR imaging at day 30 post-tumour cell implant. By day 37, all control-treated mice had died, whereas vaccine-treated mice cleared their tumours with no tumour burden detectable by histology at day 150 (N= 5 per group, **p= 0.0023). In a follow-up experiment with vaccine or control treatment on days 10, 14, 18, and 25 post-cell implants, all control mice had died by day 39. Further, vaccine-treated surviving animals were re-challenged with tumour cell implant into the contralateral hemisphere on day 42 and remain alive as of day 90. Our ongoing proof-of-principle work demonstrates that neoantigen driver mutations in high-grade glioma models can be targeted with an mRNA lipid vaccine. We hope this pre-clinical work will lead to early-phase clinical trials.

CSIG-23. CYTOMEGALOVIRUS-INFECTED HUMAN GLIOBLASTOMA CELL LINES SHOW AN AGGRESSIVE PHENOTYPE ASSOCIATED WITH UPREGULATION OF IL-6/STAT3 AND AKT SIGNALING

Abstract BACKGROUND Cytomegalovirus (CMV) has been implicated in glioblastoma (GBM) pathogenesis by affecting stemness, angiogenesis and immune pathways. Clinical trials targeting CMV in GBM patients have shown some early promise, and CMV seropositivity has been associated with poorer outcomes. However, the underlying mechanisms remain unclear. Here we investigated the effects of CMV infection on critical signaling pathways in GBM (IL6/STAT3, and Akt signaling) based on the hypothesis that CMV may contribute to poorer outcomes in GBM by increasing oncogenic signaling. MATERIALS AND METHODS Human U251 and LN229 GBM cell lines were infected with mCherry expressing human CMV TB40 strain at a multiplicity of infection (MOI) of 0.5. We performed basic phenotypic studies in vitro and investigated the status of IL6/STAT3, and Akt signaling by Western blotting. RESULTS Both LN229 and U251 cell lines were readily infectable by CMV. Infection of U251 cells was over 90% and in LN229 cells was approximately 60%. Interestingly expression of CMV was sustained over many passages in these cell lines. Infection was also verified by the presence of the CMV pp65 protein assessed by Western blotting. Measurement of cell proliferation indicated faster growth after CMV infection. Interrogation of key pro-oncogenic signaling pathways in GBM revealed a robust upregulation of IL6, as well as phospho(Tyr705)-STAT3 and phospho(Thr308)-Akt. This elevation of pro-oncogenic signaling pathways was observed in both cell lines compared with controls. CONCLUSIONS Here we show for the first time that infection of human GBM cell lines with CMV results in a more aggressive phenotype associated with upregulation of IL6/STAT3 signaling as well as a significant increase in phospho-Akt levels. We are currently studying sensitivity to standard therapies and additional molecular changes in these cells. These data support the hypothesis that CMV causes more aggressive disease and that CMV is a relevant therapeutic target in GBM.

CSIG-25. MAPK/ERK SIGNALING IN GLIOMAS MODULATES INTERFERON RESPONSES, PROMOTES TUMOR-MICROGLIA CROSSTALK, AND CONFERS SUSCEPTIBILITY TO ANTI-PD-1 AND ANTI-CTLA-4 IMMUNOTHERAPY

Abstract While immune checkpoint blockade (ICB) is not effective in unselected glioblastoma (GBM) patients, we previously established that MAPK/ERK signaling is associated with overall survival following anti-PD-1 and anti-CTLA-4 treatment in recurrent GBM. However, the causal relationship between MAPK/ERK signaling and susceptibility to ICB, as well as the mechanisms underlying this association, remain to be determined. We conducted in vivo kinome-wide CRISPR/Cas9 screenings in murine gliomas to identify key regulators of susceptibility to anti-PD-1 and CD8+ T-cell response and performed survival studies to validate the most relevant hits. Additionally, paired scRNA-seq with p-ERK staining, spatial transcriptomics, and ex-vivo slice culture of BRAFV600E mutant GBM tumors treated with BRAFi/MEKi were used to determine the causal relationship between MAPK signaling, tumor cell immunogenicity, and modulation of microglia phenotype. Our CRISPR/Cas9 screening results identified the MAPK pathway, particularly the RAF-MEK-ERK pathway, as the most critical modulator of glioma susceptibility to anti-tumoral immunity across all kinases. Survival studies showed that mice bearing high p-ERK gliomas exhibited prolonged survival with anti-PD-1 treatment, which was not seen when ERK phosphorylation diminished. MAPK activation in murine gliomas led to durable anti-tumoral immunity upon implant re-challenge, with memory T cell infiltration in long-term survivors. Elevated p-ERK in glioma cells was associated with interferon responses and antigen presentation. Moreover, in BRAFV600E human GBM cells with ERK1/2 knockout and in slice cultures of human BRAFV600E GBM tissue analyzed by spatial transcriptomics, we observed modulation of interferon responses by the MAPK/ERK pathway. Notably, in slice cultures from BRAFV600E mutant GBM, BRAFi/MEKi treatment disrupted the interaction between tumor cells and tumor-associated macrophages/microglia. In conclusion, the MAPK/ERK pathway is a critical driver of GBM susceptibility to anti-tumoral immunity, modulating the interferon responses and antigen-presenting machinery in glioma cells, as well as tumor cell interaction with the microenvironment.

IMMU-26. TARGETING CHK2-YBX1/3 INTERACTION TO OVERCOME IMMUNOSUPPRESSION IN GLIOMAS

Abstract Whereas the contribution of tumor microenvironment to the profound immune suppression of glioblastoma (GBM) is clear, tumor-cell intrinsic mechanisms that regulate resistance to CD8 T cells are less studied. We performed an in vivo CRISPR screen to identify glioma intrinsic kinases that contribute to evasion of tumor cells from CD8 T cells. The screen revealed a DNA damage response gene-checkpoint kinase 2 (Chek2) to be the most important kinase contributing to escape from CD8 T-cell recognition. Genetic depletion or pharmacological inhibition of Chek2 with blood-brain-barrier permeable drugs that are in the clinical trials, in combination with PD-1/PD-L1 blockade, led to survival benefit in preclinical glioma models. Analysis of human GBMs demonstrated that Chek2 expression is inversely associated with antigen presentation and T-cell activation. However, the mechanism underlying the immunosuppressive function of Chek2 in gliomas is unknown. Here, we identified that glioma tumor cell intrinsic Chek2 interacts with Y-Box Binding Protein 1 and 3 (Ybx1&3) in ex vivo gliomas using immunoprecipitation followed by mass spectrometry. Independently, YBX3 was also identified as one of the most significantly phosphorylated protein upon Chek2 knockout using phosphoproteomics analysis. The YBX1&3 are DNA/RNA binding proteins implicated in transcriptional regulation of immune-modulatory genes. In silico docking using Alphafold and PIPER tool in Schrodinger revealed that the YBX1 cold-shock domain interacted near the ATP/ADP binding site in CHK2. This interface was also part of the CHK2-CHK2 dimer interface. Consistent with our model, disruption of CHK2 dimerization using prexasertib led to increased colocalization of CHK2-YBX1 in human GBM patient derived xenograft cells. Further, targeting of CHK2-YBX1 interaction with YBX1 inhibitor SU056 reversed the repression of IFNγ response genes in glioma cells. Together this study unveils a kinase independent (non-catalytic) function of CHK2, providing insight into the non-canonical immunosuppressive role of CHK2 in gliomas.

TMIC-66. A NEW MOUSE MODEL THAT COMBINES DRIVER MUTATIONS TO ACCURATELY RECAPITULATE THE DIVERSE PHENOTYPES OF HUMAN GLIOBLASTOMA

Abstract To effectively beat glioblastoma, new pre-clinical models are needed: to dissect the biology of processes like migration and therapy resistance; to understand immune suppression and immunotherapy; and to use as a model for testing in therapeutic development. We have developed a new panel of glioblastoma mouse models, which engineer well-characterised glioblastoma driver mutations into an immortalised eGFP murine astrocyte-like cell. These “GEM-CLeM” are Genetically Engineered Mouse Cell Line Models. When transplanted intra-cranially into immunocompetent mice, these lines formed tumors with the key characteristics of glioblastoma – migratory and invasive, with rapid proliferation and infiltration of suppressive immune cells. The eGFP expression allowed detection of glioblastoma cells in situ and ex vivo, and the ability to readily distinguish tumor and stromal components of the tumors. Importantly, different combinations of driver mutations led to diverse tumor phenotypes. Loss of Pten with over-expression of mutant RAS generated tumors with 100% efficiency and the same time to tumor onset as the commonly used GL261 cell line. However, unlike GL261 tumors the Pten-RASV12 tumors had a highly invasive edge and extensive migration, and were full of pro-tumorigenic macrophages and monocytes, with very few T-cells or dendritic cells. Together, this recapitulated the phenotype of human wildtype IDH1 glioblastoma. In contrast, the very common EGFRvIII mutant did not drive tumor formation in combination with Pten loss. We also modelled mutant IDH glioblastoma by combining loss of p53 with overexpression of IDH1R132H. These were slow growing, densely packed tumors with extensive microglial involvement in addition to pro-tumorigenic macrophages. This GEM-CLeM system combines the benefits of the traditional genetically engineered mouse models with accessible, rapid, reproducible tumor formation. The GEM-CLeM lines are amenable to further genetic manipulation, so cells can be customised with other mutations to ask important biological questions in the presence of an intact tumor immune microenvironment.

TMIC-21. PYK2/MAPK/ERK SIGNALING PATHWAY REGULATES EXTRACELLULAR VESICLE RELEASE IN GLIOBLASTOMA

Abstract Glioblastoma (GBM), the deadliest brain cancer, typically proves fatal within a year despite treatment. Tumor-associated myeloid cells (TAMs) support GBM growth, treatment resistance, and immune surveillance. Extracellular vesicles (EVs) facilitate communication between tumor and TAMs, exchanging proteins, lipids, RNA, and genetic material. As EVs can travel through the bloodstream to distant tissues, they trigger signaling pathways and metabolic changes in remote organs while also recruiting bone marrow-derived cells and modulating immune responses. Therefore, EVs serve as a crucial mechanism for the distant regulation of the tumor’s immune microenvironment. We hypothesize that Pyk2/MAPK/Erk signaling regulates EV release by modulating the actin cytoskeleton, impacting TAM infiltration and activation in tumors. This study aims to investigate the role of Pyk2/MAPK/Erk in specific EV release in GBM cells, potentially offering insights into therapeutic targets. Using confocal imaging and flow cytometric analysis of EVs, purified from medium conditioned from human primary GBM cell lines with and without Pyk2 CRISPR/Cas9 knock-out (Pyk2KO), the study identified that Pyk2KO cells do not release the population of EVs with diameter bigger then 600nm and up-regulate release of smaller EVs. Additionally, flow cytometric analysis of EVs, released from cells, fluorescently labeled with membrane dye carboxyfluorescein diacetate succinimidyl ester (CFDA-SE), detected 14% of CFSE+ events in EV’s population, purified from Pyk2WT, vs. to 2.5% from Pyk2KO cells. Membrane labeling of cells enables the tracking of microvesicles (MV) shedding and was used to differentiate between MV and ES based on the process of their biogenesis. Considering that larger size and the transition of fluorescence from the plasma membrane are indicative of MVs, our data indicate that Pyk2 is involved in the regulation of MV release. FUNDING: PRSTRT2022 and NIH Grant 1R15CA287203

STEM-14. INTEGRATED ANALYSIS OF GLIOBLASTOMA PROGENITOR SUBTYPES REVEALS A TUMOR-DERIVED STEM CELL POPULATION WITH NEURAL AND VASCULAR POTENTIAL

Abstract Glioblastoma (GBM) exhibits a vast heterogeneity in cell types and states. Recent research highlights the reactivation of neurodevelopmental programs in GBM, contributing to this heterogeneity and, ultimately, therapy resistance. To characterize the landscape of progenitor cell types in human primary GBM, we compiled a meta-atlas of single-cell transcriptomes from seven published studies. Using a novel bioinformatic method for gene program analysis, we derived GBM gene expression meta-modules represented across multiple tumors. Our analyses validated previously described GBM gene programs and revealed novel tumor progenitor subtypes. Among these subtypes, we identified a tumor-derived stem cell population exhibiting a mixed vascular identity and transcriptional programs reminiscent of neural stem cells, which we identified as a putative neurovascular progenitor (NVP). To our knowledge, this population has not been previously described in GBM. We validated the existence of NVPs in situ using immunofluorescence from primary patient GBM samples. We then performed an enrichment for NVPs using fluorescence-activated cell sorting direct from patient tumors, followed by single-cell RNA sequencing. Examination of data from traditional model systems, including gliomaspheres and mouse GBM models, confirmed the preservation of NVP identity across systems. In vivo analysis of NVPs in multiple mouse models of GBM elucidate the influence of NVPs on GBM cell type composition, highlighting their role as multipotent progenitors. Finally, we utilized single-cell lineage barcoding to trace the progeny of patient-derived NVP cells in a novel organoid transplantation model, revealing NVP differentiation into vascular-like and neural-like cells. These data indicate that NVP cells play crucial roles in tumor plasticity and may represent a valuable therapeutic target for future exploration.

EXTH-58. BLOCKING IMMUNE CHECKPOINT— LAIR1: DISRUPTING THE LAIR1—FXIII-A—COLLAGEN IMMUNOSUPPRESSIVE CIRCUIT FOR ENHANCED ANTITUMOR THERAPEUTICS

Abstract BACKGROUND Recent evidence indicates that tumor-associated myeloid cells (TAMCs), including tumor-associated macrophages (TAMs) and myeloid-derived suppressor cells (MDSCs), play a crucial role in glioblastoma (GBM) immunosuppression and progression. These cells can constitute up to 50% of the total mass in GBM tumors and are pivotal in dampening the immune response, which negatively impacts patient survival. Targeting TAMCs represents a promising strategy to overcome the limitations of current cancer treatments. However, drug development in this area remains limited. Our study focuses on Leukocyte-Associated Immunoglobulin-like Receptor 1 (LAIR1), a novel immune checkpoint overexpressed on TAMCs within GBM. We comprehensively analyze LAIR1’s inhibitory role in cancer progression, highlighting its potential as a therapeutic target. METHODS Both in vitro and in vivo experiments were conducted. LAIR1 wild-type (LAIR1+/+) and knock-out (LAIR1-/-) tumor models were tested for antitumor response and intratumoral immune landscape. We also utilized 2D and 3D cultures to evaluate the effects of antibody blockade (aLAIR1) alone and in combination with CAR-T therapy (8R-70CAR) in both human and mouse GBM models. RESULTS We have uncovered a previously unrecognized immunosuppressive LAIR1→ Factor XIII A (FXIII-A)→ Collagen IV circuit across cancer types. Inhibiting LAIR1, either through genetic LAIR1-/- or aLAIR1, effectively disrupts this immunosuppressive pathway and results in enhanced peripheral and tumor-infiltrating memory CD8 T cell populations, a phenotypic shift of TAMs towards an M1 profile, and a decrease in tumor collagen deposition. These collective effects contribute to the normalization of the TME and facilitate improved interactions between T cells and tumor cells, leading to a more effective antitumor response. Notably, using aLAIR1 as a standalone intervention or combined with CAR T cell therapy demonstrates enhanced antitumor efficacy in preclinical models resistant to chemo-radiation and anti-PD-1 treatments. CONCLUSIONS We discovered a novel mechanism by which LAIR1 contributes to GBM progression. LAIR1 blockade holds promise in reversing suppressive TME and serving as a therapeutic strategy.

IMMU-22. TWIST1-MEDIATED ENDOTHELIAL PLASTICITY DRIVES GLIOBLASTOMA RESISTANCE TO CAR T IMMUNOTHERAPY

Abstract Glioblastoma (GBM) is the most common and most aggressive malignant primary brain tumor in adults. Among the most lethal human malignancies, GBM is highly resistant to cytotoxic treatments and molecularly targeted therapies, and also generally refractory to T cell–based immunotherapies, largely due to their immunologically cold nature, i.e., characterized by a paucity of tumor T cell infiltrates that result from the immunosuppressive microenvironment. Here, we identify a distinct mechanism driven by spatiotemporal interaction of leukocytes with tumor stromal cells, namely, mesenchymal-like population of endothelial cells (ECs) form an immunosuppressive vascular niche that regulates macrophage polarization and induces GBM resistance to CAR T immunotherapy. Our single-cell transcriptome analyses of human and mouse GBM tumors identify a subpopulation of ECs acquire robust immunosuppressive phonotypes to drive alternative macrophage polarization. These ECs are spatially localized proximately to immunosuppressive macrophages and selectively express Twist1. Furthermore, we reveal a Twist1/SATB1-mediated sequential transcriptional activation mechanism, through which Twist1+ tumor ECs produce osteopontin to locally promote immunosuppressive macrophage phenotypes. Genetic or pharmacological ablation of Twist1 reverses macrophage-mediated immunosuppression and enhances T cell infiltration and activation, leading to reduced GBM growth and extended mouse survival. Importantly, pharmacological inhibition and lipid nanoparticle-mediated targeted inactivation of Twist1 overcome GBM resistance to Egfrviii CAR T cell immunotherapy. Thus, these findings uncover a spatially restricted mechanism controlling vasculature-mediated tumor immunity and suggest that targeting endothelial Twist1 may offer exciting opportunities for GBM immunotherapy.

DDDR-03. DEVELOPMENT OF ANTI-TUMOR AROMA THERAPY FOR GLIOBLASTOMA WITH LEMONGRASS ESSENTIAL OIL

Abstract INTRODUCTION Aroma essential oils apply to cancer therapy because of their immune-enhancing and sedative effects, and their ability to reduce the side effects of chemotherapy. Recently, aroma essential oils have also been reported to have anti-tumor effects. We investigate the anti-tumor effects of aromatic compounds contained in essential oils as a novel treatment for glioblastoma. In this study, we analyzed the antitumor effect of lemongrass essential oil and investigated its antitumor aroma therapy for glioblastoma. methods and RESULTS Human glioblastoma cells (U87, T98G) and mouse brain tumor cells (RSV-M) were used in the study. Tumor cells were cultured in the presence of lemongrass essential oil for 72 hours, and the growth inhibitory effect was observed, suggesting that the evaporated lemongrass essential oil components acted on tumor cells in the vicinity. In addition, a similar effect was observed with citral alone, which is the main component of lemongrass. Analysis of the effects of citral revealed that citral strongly binds to MARK4 and inhibits its enzyme activity, and that citral decreases the phosphorylation of tau protein. Next, a mouse brain tumor model with RSV-M was established, and the anti-tumor effect of lemongrass essential oil was studied. The results showed that the mice that inhaled lemongrass essential oil for 12 h/day had a significantly prolonged survival period compared to the control group. DISCUSSION Citral is the main component of lemongrass essential oil, which contains 70-80% of lemongrass essential oil. This study suggests a mechanism of inhibition of tumor cell proliferation by citral in malignant gliomas. Citral is a volatile monoterpene, and its anti-tumor effect was demonstrated by diffusion through evaporation. CONCLUSION Citral in lemongrass essential oil shows anti-tumor effects and is expected to be applied to a new glioblastoma therapy using “aroma”

IMMU-24. GLIOBLASTOMA ENRICHED GLYCOSPHINGOLIPIDS MODULATE THE FUNCTION OF HUMAN INKT CELLS

Abstract Glioblastoma is an aggressive primary brain tumor highly resistant to currently available immunotherapies. A deeper understanding of its underlying immunoregulatory mechanisms is paramount to developing future immunotherapeutic treatments. Invariant natural killer T (iNKT) cells are unconventional T cells that recognize lipid antigens presented by an MHC-like molecule called CD1d. Although iNKT cells have been shown to regulate tumor immunity in other cancer types, their role in glioblastoma is not well characterized. Given the lipid-rich nature of the brain and the unique metabolic activity of glioblastoma cells, we hypothesized that interactions between glioblastoma and iNKT cells through glioblastoma-produced lipids could contribute to the immunosuppressive nature of the disease. We report multiple lipid species enriched in aggressive human glioblastoma stem-like cell (GSC) lines that activate human iNKT cells and modulate their functions. Lipidomic LC-MS analysis of GSCs, low-grade glioma stem-like cell lines, and normal human astrocytes revealed various glycosphingolipid species, including sulfatides, highly enriched in the GSCs. Multiple enriched sulfatide species, when presented by CD1d, were recognized by and activated iNKT cells in a dose-dependent manner. Additionally, cytokine analysis of stimulated human PBMC-derived iNKT cells demonstrated that the enriched sulfatides did not induce Th1 cytokine production, but rather many non-Th1 cytokines that potentially counteract Th1-type immune responses. This modulation of iNKT cell function by glioblastoma-enriched glycosphingolipids may contribute to the immunosuppression of glioblastoma and could highlight sulfatide production as a potential therapeutic target for glioblastoma treatment.

DDEL-18. OPTIMIZED METHOD TO QUANTIFY TTFIELDS-INDUCED PERMEABILIZATION OF CELL MEMBRANES

Abstract Tumor Treating Fields (TTFields) therapy is FDA-approved for glioblastoma. Using plate-based readouts of FITC-dextran fluorescent probes in U87 human glioblastoma cells, we previously reported increased uptake of FITC-dextrans (4-20kDa) post-TTFields exposure. However, this approach is unable to distinguish FITC-dextran uptake in live-vs-dead cells post-TTFields exposure. We demonstrate flow cytometry (FC) as a more robust and economical method to quantify the amount of FITC-dextrans entering the cells. We first identified the optimal [FITC-dextran] for the FC-based approach (titration 0-6.5mg/mL). Cells were then seeded onto coverslips and exposed to 200kHz TTFields, 1-3V/cm peak-to-peak (inovitro systemTM, Novocure), or no-TTFields (control), for 24-72h. Cells were harvested and stained for 1h using 4kDa or 20kDa FITC-dextrans. Cells were washed and analyzed by FC for mean±SD fluorescence intensity, and compared by unpaired t-test. Dead cells were excluded using propidium iodide staining. In the control vs. TTFields group, 4kDa FITC-dextrans had significantly increased uptake at 24h (4.9±0.3 vs. 8.0±1.7, p=0.01), 48h (6.5±0.2 vs. 8.3±1.0, p=0.01), and 72h (5.7±0.3 vs. 8.8±1.0, p=0.001). Likewise, in the TTFields group, the 20kDa FITC-dextrans had significantly increased uptake at 24h (5.6±0.3 vs. 7.5±0.9, p=0.006), 48h (4.8±0.3 vs. 6.6±0.8, p=0.006), and 72h (5.3±0.2 vs. 11.3±2.6, p=0.004). In the TTFields group, the 20kDa FITC-dextrans had significantly increased uptake between the 24h and 72h timepoints (p=0.03). The FC-based method utilized 0.36mg of FITC-dextrans/sample, much less compared to the 1-3mg recommended for plate-based methods. Another advantage of the FC-based method was exclusion of the dead cell population after TTFields exposure. We validated a FC-based-method that is economical and can reproducibly quantify the membrane permeabilization induced by TTFields. Compared to control conditions, TTFields increase membrane permeabilization of cells that remain alive after TTFields exposure, in a time-dependent manner for the 20kDa probe.