Cell Culture Research Articles

A guide for blood-brain barrier models.

Understanding the intricate mechanisms underlying brain-related diseases hinges on unraveling the pivotal role of the blood-brain barrier (BBB), an essential dynamic interface crucial for maintaining brain equilibrium. This review offers a comprehensive analysis of BBB physiology, delving into its cellular and molecular components while exploring a wide range of in vivo and in vitro BBB models. Notably, recent advancements in 3D cell culture techniques are explicitly discussed, as they have significantly improved the fidelity of BBB modeling by enabling the replication of physiologically relevant environments under flow conditions. Special attention is given to the cellular aspects of in vitro BBB models, alongside discussions on advances in stem cell technologies, providing valuable insights into generating robust cellular systems for BBB modeling. The diverse array of cell types used in BBB modeling, depending on their sources, is meticulously examined in this comprehensive review, scrutinizing their respective derivation protocols and implications. By synthesizing diverse approaches, this review sheds light on the improvements of BBB models to capture physiological conditions, aiding in understanding BBB interactions in health and disease conditions to foster clinical developments.

Abstract 4116603: Interleukin-33 from Necrotic Tunica Media Plays a Key Role in the Exacerbations of Coronary Arteritis in Kawasaki Disease

Objective: Alarmins resulting from cell death or oxidative stress have been shown to be involved in the development of Kawasaki disease (KD) vasculitis. In our previous study, we demonstrated the potential role of (IL)-33 as an alarmin in the development of KD vasculitis. Although edematous dissociation (necrotic change) of the tunica media is thought to be a major source of IL-33 in KD vasculitis, it has not yet been elucidated. Methods: We investigated the impact of IL-33 released from necrotic human coronary artery smooth muscle cells (HCASMCs) on human coronary artery endothelial cells (HCAECs) by a co-culture assay using the Transwell ® cell culture insert system. Subsequently, we evaluated the anti-inflammatory effects of anti-IL-33 and anti-ST2 antibodies compared to the conventional therapies of KD, such as high-dose IgG or anti-tumor necrosis factor (TNF)-α antibody. Results: Primary necrosis of HCASMCs induced significant release of IL-33. In co-cultures of necrotic HCASMCs with HCAECs, necrotic HCASMCs significantly induced the production of various proinflammatory cytokines in HCAECs. Anti-IL-33 and anti-ST2 antibodies exhibited unique inhibitory effects on the production of platelet-derived growth factor-BB or IL-12(p70) in HCAECs. Conclusion: The results of the present study suggest the potential involvement of edematous dissociation of the tunica media in the development of KD vasculitis. Furthermore, the distinctive anti-inflammatory effects of the anti-IL-33/ST2 axis drugs suggest novel therapeutic options for patients with refractory KD.

Assessing Drug Sensitivity in Fission Yeast Using Half-Maximal Inhibitory Concentration (IC50) Assays.

Fission yeast is an excellent model organism in which to study mammalian drug sensitivities. In addition to building a mechanistic picture of drug effect, fission yeast screens may be valuable in determining compounds that show synthetic lethality effects. While compounds might be screened for a variety of phenotypes, an effective method is to detect the proliferative effects of a new compound on a yeast culture. This is traditionally performed in acute viability assays or spot tests; both methods require some knowledge of concentration to observe an effect. Mammalian cell culture experiments that assess proliferation to indicate drug dose and effect are well described. However, differences between S. pombe growth characteristics and mammalian cells mean that importing a mammalian viability assay requires consideration of potential effects on fission yeast biology. We describe the half-maximum inhibitory (IC50) dose as a method of rapid proliferation effect screening. IC50 determination is performed on liquid cultures in 96-well plates and may be developed for initial compound library uses or in synthetic lethality screening of drug effects.

EXTH-71. A MECHANISTIC INVESTIGATION INTO AUGER THERAPY: 2-DIMENSIONAL THIN-FILMS TO CHARACTERIZE THERAPEUTIC RANGEIN VITRO

Abstract Metallic nanoparticles can improve the accuracy and effectiveness of conventional radiotherapy in the treatment of solid tumors through a light-matter interaction known as the Auger effect. In this work, we present the assembly of an iron oxide (Fe3O4) nanoparticle (NPs) layer within a biocompatible polyelectrolyte film using ionic self-assembly to enable the spatial separation of NPs from their target cells. Films exhibited uniform nanoparticle coverage as well as strong stability in cell culture conditions. To investigate the applicability of NMDR with non-internalized NPs, conditions with (NP-PEM) and without (PEM) embedded nanoparticles were used to evaluate the therapeutic effect of physically separated nanoparticles on cells cultured on the film surface via a measure of intracellular reactive oxygen (ROS) generation and histone phosphorylation (yH2AX) after irradiation from a 137Cesium source. Cells cultured on NP-PEM demonstrated a 78.4% increase in ROS generation when compared to cells cultured on PEM films. When adjusting for cell count, NP-PEM films induce 70.5% greater ROS generation. In investigating nanoparticle-mediated deposition of radiation (NMDR), these films allow for the determination of the extent of the therapeutic effect when NPs are not internalized into their targeted cells, potentially providing indications with respect to off-target effects. Cells were irradiated on films with nanoparticle distances from 25.9 nm to 97.5 nm to interrogate the NMDR effect size at a range of distances. yH2AX phosphorylation exhibited an exponential decay until 69.8 nm, where there was no difference when compared to nanoparticle-free films. This work highlights the ability of iron oxide NPs to deliver NMDR at a known, quantifiable distance, providing insights for further translational applications.

PATH-64. UNDERSTANDING MECHANISTIC UNDERPINNINGS OF MOLECULAR SUBGROUPS OF MENINGIOMA

Abstract Meningiomas are the most common intracranial tumors. In the past, they were thought to be indolent and could be treated by resection. The current standard of care utilizes WHO histopathological grading to identify malignant potential. Our lab identified three molecular groups (MenG A, B, and C) using CNVs, methylation, and/or expression profiling with different biological characteristics. These groups were independently validated and predicted recurrence more reliably than WHO grade. MenG A is genomically stable but harbors tumors with point mutations, mainly in TRAF7, KLF4, and AKT1. On the other hand, MenG B and C tumors have chromosome 22q arm deletion or loss of merlin, the protein product of the NF2 gene. Moreover, MenG B is characterized by DNA hypomethylation, while MenG C tumors show DNA hypermethylation and have additional chromosomal losses, most notably in chromosome 1p. Lastly, while MenG A/B are indolent, MenG C – comprising approximately 28% of meningiomas – recur frequently and are refractory to radiotherapy and/or surgery. How can two groups of tumors with the same driver mutation behave so differently? Hence, our lab is interested in (1) what molecular pathway/factor(s) are necessary for becoming any meningioma, and (2) what mechanisms are critical for the aggressiveness of MenG C. Here, we investigate the role of TRAF7 in meningioma biology. Toward this end, we study interactors, modulators, and individual mutations in cell culture. In addition, we use published exome sequencing and expression data to reveal possible connections between chromatin state and individual genes. In conclusion, a variety of underlying mechanisms drive meningioma tumorigenicity and aggressiveness. Elucidating these mechanisms could provide future therapeutic targets for currently untreatable meningiomas.

CCRG-03. DETECTION OF HETEROGENEOUS CELL CYCLE ALTERATIONS UPON TMZ CHEMOTHERAPY TREATMENT THROUGH ADVANCED MACHINE LEARNING

Abstract Glioblastoma (GB), the most aggressive central nervous system tumor, is hallmarked by its frequent relapse under alkylating chemotherapy. GB cells frequently exhibit changes in cell cycle phases during TMZ treatment, with a reduction in the G0-G1 phase and an increase in the S and G2 phases. This indicate both tumor cell re-entry into the cell cycle and a TMZ-induced arrest, contributing to resistance and progression. In this study, we leverage dynamic protein abundance of Ki-67, a proliferation marker peaking during S, G2, and M phases, to analyse cellular responses to TMZ in various cell lines and tissue samples. We designed an image-based machine learning approach for pattern recognition to reconstruct and analyse the cell cycle at the single-cell level. Six primary cell lines were characterized through whole-genome DNA, methylation and RNA sequencing. Functional read-outs were performed with large-scale, time- and concentration-dependent TMZ treatments in cell culture and GB-tissue slice models. Our model successfully reconstructed detailed cell cycle stages, showing that untreated cells were predominantly in the G0-G1 phase. TMZ treatment significantly shifted cells toward the S/G2 phase (p < 0.02) in 3 of 6 cell lines and the pseudo-time reconstruction highlighted dose-dependent cell cycle changes. Cell lines with enhanced stem-like features, defined based on epigenetic and transcriptional data signature, showed no cell cycle changes at high TMZ doses (>150µM), regardless the MGMT promoter methylation status. This heterogeneity in cell cycle response emphasizes the complexity of GB and the need for personalized treatments. Our study provides insights into GB MGMT cell cycle alterations and resistance mechanisms that are independent of MGMT methylation status, a known TMZ response biomarker. Therefore, the observed TMZ-induced cell cycle changes may serve as novel biomarkers for assessing treatment response, paving the way for personalized therapeutic approaches.

DDDR-40. TARGETING BCL-XL WITH MIRZOTAMAB CLETUZOCLAX (ABBV-155) AND STANDARD OF CARE THERAPIES OVERCOMES APOPTOTIC RESISTANCE IN GLIOBLASTOMA

Abstract Conventional GBM therapies (e.g., temozolomide (TMZ), irradiation (IR)) transiently halt tumor growth of glioblastoma (GBM) but fail to induce apoptosis, inevitably leading to disease progression and poor patient survival. Targeting apoptotic blocks with BH3 mimetics can be an efficacious strategy to trigger apoptosis; however, these therapies can prevent high exposures in patients due to on-target, off-tumor toxicity in normal tissues. Mirzotamab Cletuzoclax (ABBV-155) is a first-in-class antibody-drug conjugate (ADC) that is specific for the highly expressed cell surface protein B7-H3 (CD276) and delivers a selective and potent BCL-XL inhibitor warhead. Here we identified that both TMZ and IR leave p53 WT GBM tumors exclusively dependent on the apoptotic block, BCL-XL, for survival. ABBV-155 negated the BCL-XL apoptotic block in both cell culture and orthotopic xenograft GBM models. Consequently, ABBV-155 combined with TMZ or IR initiates apoptosis and is synergistically lethal in GBM cells. Moreover, these combinations decrease tumor burden and extend survival in patient derived orthotopic xenografts. Taken together, these results demonstrate that the combination of DNA damaging therapy and ABBV-155 can have a synergistic anti-tumor impact in p53 WT GBM.

Fibrillar Hydrogel Inducing Cell Mechanotransduction for Tissue Engineering.

One of the key strategies for tissue engineering is to design multifunctional bioinks that balance printability with cytocompatibility. Here, we describe fibrillar hydrogels produced by Schiff base formation between B-type gelatin and oxidized sodium alginate, followed by the incorporation of type I collagen, yielding a new gel (MyoColl). The resulting hydrogel exhibits a temperature- and mass-ratio-dependent sol-gel transition, showing variability of hydrogel properties depending on the component ratio. MyoColl composition provides a convenient platform for biofabrication in terms of shear thinning, yielding, Young's modulus, and shape accuracy. Metabolic activity tests and fluorescent microscopy of 2D hydrogel-based mouse C2C12 myoblast cell culture show significant cytocompatibility of the developed carriers. In addition, primary signs of cell mechanotransduction and myofilament formation of 3D printed MyoColl-based cell cultures were detected and described. Due to these promising results, the described hydrogel composition has shown itself as a convenient platform for muscle tissue engineering.

DDDR-48. EZH2 INHIBITION AS A POTENTIAL THERAPEUTIC AVENUE IN THE TREATMENT OF MENINGIOMA

Abstract INTRODUCTION High grade meningiomas provide challenges to treatment as these neoplasms are more likely to recur, exhibit invasion of brain parenchyma, and result in decreased long term survival. Recent works demonstrate the epigenetic modifier, EZH2, to be increased in higher grade meningiomas, and to correlate with poor survival. The selective targeting of EZH2 has shown promise in other malignancies with overexpression of or gain-of-function mutations in EZH2, leading to promising findings. Here we sought to investigate the effect of EZH2 inhibition on meningioma cell growth and viability, using a panel of in vitro models. METHODS Human immortalized meningioma cell lines, mouse meningioma cell lines and a panel of human primary cell cultures with varying baseline protein expression of NF2, SMARCB1, and EZH2, were selected for investigation. Cells were treated with several selective small molecule inhibitors of EZH2. Cell proliferation, cell cycle and cell death processes were assessed. RNAseq was used to assess transcriptional changes in response to EZH2 inhibition. Western blots were used to assess protein changes in response to EZH2 inhibition. RESULTS EZH2 inhibition significantly impairs meningioma cell proliferation, at varying IC50s, in all cell lines and primary cultures tested, utilizing a panel of EZH2 inhibitors. EZH2 inhibition resulted in significantly decreased colony formation in a high-grade cell line. Furthermore, EZH2 inhibition results in a reduction of Ki67+ cells, induction of cell cycle arrest, and apoptosis. Similar grouping of differential gene expression in immortalized cell lines and primary cell cultures is observed across cell lines and primary cell cultures, after EZH2 inhibition. CONCLUSION Our work demonstrates that EZH2 inhibition results in growth inhibition, cell cycle arrest, and cell death, across all cell lines, and cell cultures tested. Futhermore, modulation of protein levels of the EZH2 target H3K27 is observed upon treatment. RNA changes are broad and involve cell proliferative and migratory processes. This work provides the framework for further investigation of EZH2 inhibition in meningioma.

TMOD-16. ZUCLOPENTHIXOL REDUCES ACTIVITY-DEPENDENT GLIOBLASTOMA PROLIFERATION

Abstract A major challenge in modeling glioblastoma in the laboratory is the complex microenvironment in which tumors evolve in humans. The varied architecture and immune privilege of the brain limits the external validity of many experiments performed in modern cell biology laboratories. Organotypic human cortical slices present an opportunity to model glioblastoma and test the effect of drugs against glioma in ex-vivo human tissue. Cortical slice cultures maintain the cellular organization of the human brain, more closely approximating glioblastoma proliferation in patients. In this study, we test the effect zuclopenthixol, a repurposed antipsychotic inhibitor of glioblastoma-induced neuronal hyperexcitability, on the proliferation of glioblastoma cells in an organotypic human cortical slice culture. Primary patient-derived glioblastoma cultures were established, and human cortical tissues were emersed in oxygenated ACSF agarose gel before preparing 250 µm thick slices. Slices were either exogenously seeded with glioblastoma cells or stained for endogenous tumor infiltration. Slices with either endogenous or exogenous seeding were then treated with zuclopenthixol or regular media change and cultured for 10 days. Proliferation was measured with Ki67 staining and quantified via automated pixel summation using the Pillow library in Python. 0.1 µM zuclopenthixol treated slices had significantly lower proliferation index in both the endogenous tumor condition (0.04 vs 0.30, P < 0.05) and the exogenous seeding condition (0.0013 vs 0.028, P = 0.033). Reduced tissue integrity was observed beginning day 8 of culture, as tissue surrounding tumor seeding sites eroded. Here, we model glioblastoma proliferation in live human tissue and quantify the anti-proliferative effect of zuclopenthixol. We show that zuclopenthixol treatment significantly reduced the proliferation index. Our experimental model allowed the optimization of organotypic human cortical slice cultures for immunofluorescence studies and live-cell imaging.

CNSC-01. PEDIATRIC GLIOMAS EXPRESS CIRCADIAN GENES AND DEMONSTRATE CIRCADIAN REGULATION OF TEMOZOLOMIDE SENSITIVITY

Abstract INTRODUCTION Pediatric high-grade gliomas (pHGG) are highly invasive brain tumors with dismal survival rates, prompting the need for innovative therapeutic strategies. Previous research in adult glioblastoma patients revealed a correlation between the timing of Temozolomide administration and patient survival. There has, however, been limited exploration into circadian gene expression in pediatric gliomas. In this study, we investigate whether pHGGs exhibit rhythmic expression of circadian genes and whether the timing of drug administration influences Temozolomide sensitivity. METHODS A bioinformatics approach, immunocytochemistry and immunoblotting were used to assess key circadian genes in low- and high-grade gliomas. We then used timed qPCR to assess rhythmic expression in BMAL1 and REV-ERBα, two key circadian genes, across 48 hours in our pHGG cells following dexamethasone cell synchronization. Lastly, we used CyQuant Proliferation Assays to examine the effects of Temozolomide applied at peaks versus troughs in BMAL1 expression. RESULTS Using data from PedcBioPortal, we found significantly higher expression of BMAL1 in pHGGs compared to pLGGs (0.349 + 1.05 vs 0.0545 + 0.666, p < 0.001). We identified cellular localization of BMAL1 and REV-ERBα in pHGGs along with rhythmic expression of BMAL1 and REV-ERBα in our three pHGGs primary cell cultures across 48 hours (JTK_CYCLE, p < 0.005). Lastly, we demonstrated a chronotherapeutic response to Temozolomide in two of our pHGG cell lines, with significantly decreased proliferation when treatment occurred at trough versus peak BMAL1 expression (p < 0.01). CONCLUSION In this study, we found that circadian genes BMAL1 and REV-ERBα are expressed in pHGGs and expression patterns differ between pHGGs versus pLGGs. Circadian gene expression is rhythmic in pHGGs, and pHGGs can have differential Temozolomide-sensitivity based on circadian cycles. These findings suggest that circadian timing of chemotherapy may influence the efficacy of treatment, and next steps will be to explore the mechanisms underlying circadian regulation of Temozolomide sensitivity.

DDDR-32. EXPLORING ANDROGENIC MODULATION IN GLIOBLASTOMA: POTENCY AND SELECTIVITY OF NOVEL NORETHINDRONE DERIVATIVES

Abstract BACKGROUND Glioblastoma (GBM) represents the most frequently diagnosed malignant CNS tumor in adults and remains incurable. Despite the identification of several oncogenic drivers contributing to tumor formation, targeted therapy with brain-penetrant inhibitors has afforded little progress in survival outcomes. Interestingly, gender has been shown to increase the incidence of GBM, with men having roughly a 50-60% increase in incidence compared to women in the US. This statistic led to research on the involvement of and subsequent inhibition of androgen synthesis and signaling in cell culture models and tumor samples. METHODS To further investigate the potential involvement of steroidal hormones in GBM proliferation, our lab synthesized a series of norethindrone (NE) derivatives with varied molecular weights, steric bulk, and lipophilicity to modulate the androgenic activity of NE. The small library was tested in several GBM cell lines, including U87, A172, and T98G cells, using cell viability assays. RESULTS One derivative, a lipophilically modified version of NE synthesized via a one-pot click reaction, was shown to have improved potency compared with parental NE (>5-fold). The lipophilic NE derivative had IC50 value of 22 uM, 15 uM, and 35 uM in the U87, A172, and T98G cells, respectively. This molecule was tested in immortalized normal human astrocyte (NHA) cells and found to have an IC50 value of 34 uM suggesting some selectivity. DISCUSSION Given this derivative’s activity in GBM models and modest selectivity, we plan to screen it in additional GBM cell lines (LN-18 and U251) and eventually in glioma stem cell models. Future research will focus on identifying key targets involved in the mechanism of action, including the impact of lipophilic addition to the steroid nucleus and its effect on androgenic activity and downstream signaling. We will investigate these effects using RPPA and co-treatments with androgen receptor antagonists and 5-alpha reductase inhibitors.

DDDR-49. REPURPOSING OF THE ANTI-VIRAL DRUG RIBAVIRIN AS A POTENTIAL MENINGIOMA THERAPEUTIC

Abstract INTRODUCTION High grade meningiomas provide significant challenges to treatment. Recent works have demonstrated meningiomas to express higher levels of the epigenetic regulator enhancer of zeste homolog 2 (EZH2), as well as the protein eIF4E, a key player in translation. These two molecules are demonstrated targets of the well tolerated antiviral drug, ribavirin, which has recently shown promise as an anti-neoplastic agent. Based on these findings we sought to investigate the effect of ribavirin on meningioma tumorigenesis utilizing in vitro and in vivo meningioma models. METHODS Several immortalized human meningioma cell lines, three immortalized mouse meningioma cell lines, and a panel of human primary cell cultures were assessed. Cell proliferation, cell colony formation, clonogenic assay, and cell death were assessed. Furthermore, we tested ribavirin efficacy in vivo utilizing two aggressive high grade syngeneic orthotopic allograft meningioma models in FVB WT mice. RESULTS We provide evidence that ribavirin significantly impairs meningioma cell growth and proliferation, and induces cell death in vitro. Most importantly, we demonstrate that ribavirin alone significantly improves the survival of mice orthotopically implanted with MGS2 cells. Ribavirin-treated animals exhibited a significantly increased median survival (67 days) compared to controls (41 days; p=0.0009;) in an orthotopic convexity meningioma xenograft model. Furthermore, ribavirin treatment resulted in increased survival in an orthotopic skull base meningioma xenograft model. CONCLUSION Our work establishes that ribavirin is effective against meningioma in vitro and in vivo. Ribavirin treatment reduced proliferation in all cell lines tested, in both high and low grade models, induced cell death, and prolonged survival in two aggressive in vivo models, potentially via modulation of the EZH2 and eIF4E pathways. Given the lack of medical therapy for high grade meningiomas, these findings provide a framework for further investigation into ribavirin’s effects on meningioma tumorigenesis, and demonstrate ribavirin as a potential new therapeutic option in the treatment of meningioma.

IMMU-36. MULTI-OMIC CHARACTERIZATION OF A SYNGENEIC MOUSE CANCER STEM CELL MODEL OF GBM FOR PRECLINICAL APPLICATIONS

Abstract Glioblastoma (GBM) is resistant to many therapies including immunotherapies. There is an urgent need to explore the role of the microenvironment in resistance to therapy with tailored pre-clinical models. Using multi-omic approaches we describe a syngeneic cancer stem cell mouse model of GBM, with a spontaneous amplification of Igf2. We investigate whether Igf2 influences tumour and microenvironment cells to promote immunosuppression in GBM. Three cell lines were previously established from spontaneous brain tumours in C57Bl/6 Trp53+/-/Nf1+/- mice and maintained under neural stem cell culture conditions. Whole genome sequencing (WGS) was used to determine single nucleotide, copy number, and structural variation, revealing loss of both copies of Trp53 and Nf1 in all three cell lines. However only one cell line, mBT0309, developed tumors when orthotopically allografted. Analysis of genomic alterations and transcriptomics revealed that mBT309 exhibits amplification of the Igf2 loci and overexpression was confirmed at the RNA and protein level. Spatial and single cell transcriptomics showed that Igf2 is overexpressed in mBT0309 allografted tumours and correlated with specific transcriptomic programs. A high-parameter Imaging Mass Cytometry (IMCTM) panel was used for spatial proteomic analysis, to monitor the development of tumours in a time-course experiment. In vitro growth characteristics and stem marker expression in both transcriptomic and spatial IMC data suggest that high levels of Igf2 may regulate GBM stemness features. Analysis of human GBM datasets revealed that Igf2 amplified tumours have reduced CXCL11 expression. These findings suggest that high levels of Igf2 may influence immunosuppression by reducing T-cell recruitment. This syngeneic immunocompetent GBM stem cell model harbouring Igf2 amplification adds to the suite of models to study the GBM microenvironment and its role in immunosuppression and resistance to therapy.

EXTH-39. A BENCH-TOP MODEL FOR THE OPTIMIZATION OF ULTRASOUND PARAMETERS FOR SONODYNAMIC THERAPY OF GLIOBLASTOMA

Abstract BACKGROUND In-vitro studies have demonstrated that low frequency sound waves can interact with hemo-porphyrin molecule to induce the production of reactive oxygen species. This biophysical phenomenon can induce tumour cell death in glioblastoma cells in-vitro. The purpose of this study was to evaluate the production of reactive oxygen species and expression of apoptotic markers in glioblastoma cells in response to varying total treatment time and ultrasound power using a bench-top focused ultrasound (FUS) device. METHODS U251 cells were exposed to 5-Aminolevulenic Acid (5-ALA) at varying concentrations for up to 24 hours. The time point with peak proto-porphyrin IX (PPIX) fluorescence was established using fluorescence activated cell sorting (FACS). A transducer utilized at 0.91MHz was placed on an inverted stage with the focus targeted to the bottom of a standard 96-well cell culture plate. Pressure wave delivery was confirmed using a hydrophone. GBM cells were treated with 5-ALA alone, FUS alone, or 5-ALA + FUS and the cells were incubated for 24 hours prior to measuring ROS induction and cell death. ROS was measured using FACS with the CellROX Green Kit for Oxidative Stress Detection and Annexin-V was measured using the Dead Cell Stain Kit. RESULTS Peak fluorescence intensity for PPIX in the U251 cell line was found to be induced through incubation with 5-ALA for 24 hours with a concentration 200mg/mL. The fluorescence intensity of PPIX was found to be 313% greater in the treated group compared to the control group. Peak ROS was observed using a 0.91MHz transducer frequency for a total time of 120s with 6W average power. ROS was 61% and 66% greater in the SDT group compared to the FUS and 5-ALA group respectively. CONCLUSION Using this bench-top set up we successfully induced ROS and apoptosis in U251 cells. Establishing a reliable in-vitro model will allow us to further investigate the effects of other sonication parameters such as burst length.

TMOD-28. MODELING MICROENVIRONMENTAL DYNAMICS: A NOVEL 3D GLIOMA-NEURON FUSION MODEL

Abstract Gliomas engage in bidirectional interactions with their microenvironment, wherein neuronal activity influences glioma proliferation and gliomas induce neuronal hyperexcitability. Although various models elucidate this complex interplay, a preference for a three-dimensional (3D) cell culture model has emerged to mimic intra-tumoral heterogeneity and microenvironmental interactions influencing glioma invasion, resistance, and recurrence. This study introduces an in vitro, self-assembled, scaffold-free, neuron-glioma spheroid fusion model to investigate the electrophysiological and functional interplay between glioma and its microenvironment. Primary mouse cortical neurons (MCN) were isolated from prenatal brain tissues and were cultured at 500,000 cells/spheroid on ultra-low attachment plates. Glioma organoids (WHO Grade 2-4) were developed from primary patient-derived tissue samples and were allowed to self-assemble for at least two weeks. After spontaneous spiking activity was detected in MCN spheroids, they were combined with glioma organoids in culture and allowed to fuse. A multielectrode array (MEA) was used to characterize the electrophysiological properties of the fusion model. Structural and functional characterizations were performed using immunofluorescence staining of proliferation, microglial, astrocytic, synaptic, and tumor markers. Additionally, the electrophysiological effects of the dual-IDH-mutant inhibitor, Vorasidenib, were assessed in WHO grade 2-4 IDH-mutant gliomas. Electrophysiological analysis of glioma-neuron co-cultures using MEA demonstrated a significantly increased network synchrony and firing rate in the fusion model when compared with the neuron-only condition, consistent with 2D models in the past. In correlation, the fusion model also demonstrated a significant increase in the Ki67 proliferation index across WHO grade 2-4 gliomas when compared to the glioma organoid-only condition. Vorasidenib treatment decreased neuronal firing rate and synchrony. Together, these results offer a high-throughput 3D model for recapitulating the tumor-brain microenvironment for both low and high-grade gliomas. Future studies will focus on the use of this model for drug screening and the exploration of malignant transformation in gliomas.

EXTH-82. TUMOR ORGANOID PREDICTED THERAPEUTIC RESPONSES TO NOVEL EPIGENETIC DRUGS VIA HIGH-THROUGHPUT SCREENING

Abstract INTRODUCTION The treatment choices for pediatric brain tumors are still very limited. Epigenetic therapy emerges as a promising option recently. The lack of in vitro models that represent tumors in vivo and can be scalable for large scale drug screening is major barrier. We have developed tumor organoids from patient-derived orthotopic xenograft and performed a multi-stage high throughput screening for single or combined epigenetic agents and radiation therapy in tumor organoids. METHOD Primary xenograft tumor cells were cultured in matrix and cancer stem cell culture medium in 384-well plates. Radiation sensitivity was examined at 5 different doses. Epigen40 (40 drugs/compounds) targeting various epigenetic modifications was used alone to identify agents with the most cell killing (4 doses for up to 19 days). The drugs that showed partial activity were combined with radiation (at 5 doses) to evaluate additive or synergistic effects. The anti-tumor efficacy of selected drug was then evaluated in vivo. RESULTS The conditions of organoid growth and screen variability were optimized. 7 tumor organoids including 4 glioblastomas and 3 medulloblastomas were successfully established and characterized. The different radio-sensitivities were observed among these models. 7 compounds (JIB-04, SP2509, AS-8351, GSK J4 HCI, 3-Deazaneplanocin A HCL, Chaetocin, TC-E-5003) were shown to effectively suppress organoid growth. Chaetocin, methyltransferase SUV39H inhibitor, demonstrated broadly active and highly potent inhibition across all 7 tested organoids. In addition, bioinformatic analysis identified a subset of epigenetic inhibitors combined with radiation that produced additive anti-tumor activities in vitro. Unfortunately, in vivo treatment of Chaetocin (0.25mg/kg) didn’t convey consistent anti-tumor effects as shown in vitro whereas a high risk of drug toxicity to the mice. CONCLUSION Tumor organoids were successfully developed from primary tumors. With our optimized assay using primary tumor organoids and a multi-stage high throughput drug screening, a novel panel of epigenetic drugs that effective alone or additively with radiation was identified.

3D-environment and muscle contraction regulate the heterogeneity of myonuclei.

Skeletal muscle formation involves tight interactions between muscle cells and associated connective tissue fibroblasts. Every muscle displays the same type of organisation, they are innervated in the middle and attached at both extremities to tendons. Myonuclei are heterogeneous along myotubes and regionalised according to these middle and tip domains. During development, as soon as myotubes are formed, myonuclei at muscle tips facing developing tendons display their own molecular program. In addition to molecular heterogeneity, a subset of tip myonuclei has a fibroblastic origin different to the classical somitic origin, highlighting a cellular heterogeneity of myonuclei in foetal myotubes. To gain insights on the functional relevance of myonucleus heterogeneity during limb development, we used 2D culture and co-culture systems to dissociate autonomous processes (occurring in 2D-cultures) from 3D-environment of tissue development. We also assessed the role of muscle contraction in myonucleus heterogeneity in paralysed limb muscles. The regionalisation of cellular heterogeneity was not observed in 2D cell culture systems and paralyzed muscles. The molecular signature of MTJ myonuclei was lost in a dish and paralysed muscles indicating a requirement of 3D-enviroment and muscle contraction for MTJ formation. Tip genes that maintain a regionalized expression at myotube tips in cultures are linked to sarcomeres. The behaviour of regionalized markers in cultured myotubes and paralyzed muscles allows us to speculate whether the genes intervene in myogenesis, myotube attachment or MTJ formation.

Navigating stem cell culture: insights, techniques, challenges, and prospects

Stem cell research holds huge promise for regenerative medicine and disease modeling, making the understanding and optimization of stem cell culture a critical aspect of advancing these therapeutic applications. This comprehensive review provides an in-depth overview of stem cell culture, including general information, contemporary techniques, encountered problems, and future perspectives. The article begins by explaining the fundamental characteristics of various stem cell types, elucidating the importance of proper culture conditions in maintaining pluripotency or lineage commitment. A detailed exploration of established culture techniques sheds light on the evolving landscape of stem cell culture methodologies. Common challenges such as genetic stability, heterogeneity, and differentiation efficiency are thoroughly discussed, with insights into cutting-edge strategies and technologies aimed at addressing these hurdles. Moreover, the article delves into the impact of substrate materials, culture media components, and biophysical cues on stem cell behavior, emphasizing the intricate interplay between the microenvironment and cell fate decisions. As stem cell research advances, ethical considerations and regulatory frameworks become increasingly important, prompting a critical examination of these aspects in the context of culture practices. Lastly, the article explores emerging perspectives, including the integration of artificial intelligence and machine learning in optimizing culture conditions, and the potential applications of stem cell-derived products in personalized medicine. This comprehensive overview aims to serve as a valuable resource for researchers and clinicians, fostering a deeper understanding of stem cell culture and its key role in advancing regenerative medicine and biomedical research.

EXTH-20. EXPLORING EUPHOL’S THERAPEUTIC EFFECT IN ADULT AND PEDIATRIC BRAZILIAN HIGH-GRADE GLIOMAS PRE-CLINICAL MODELS

Abstract High grade gliomas (HGG) are among the most aggressive brain tumors affecting both adults and children. Adult HGG (aHGG) account for almost 25% of all brain tumors in adults, with glioblastoma (GBM) being the predominant subtype. Pediatric HGG (pHGG), though less common, accounts for 3-15% of primary brain tumors in children and exhibits distinct genetic profiles compared to aHGG. Standard treatment for HGG in both adults and pediatric patients includes maximal surgical resection followed by temozolomide (TMZ)-based chemotherapy and radiation therapy. Our group previously reported that euphol, a natural compound derived from the sap of Euphorbiaceae family plants, exerts cytotoxicity effects on glioma cells. In this study, we investigated the therapeutic potential of euphol in primary cell cultures and xenograft models derived from six Brazilian HGG patients, including three adults and three pediatrics. In vitro, euphol exposure significantly reduced cell viability reduction, clonogenic capacity, and migration, while enhancing apoptosis in all six primary cultures. Additionally, euphol sensitized glioma cells to TMZ, resulting in a synergistic cytotoxic effect. In vivo, euphol reduced tumor growth in both GBM and pHGG models, and prolonged the survival of mice in the GBM orthotopic experiment. These findings highlight the promising therapeutic potential of euphol for treating high-grade gliomas in both adult and pediatric populations.