Brain Slice Cultures Research Articles

Glucose and Oxygen Levels Modulate the Pore-Forming Effects of Cholesterol-Dependent Cytolysin Pneumolysin from Streptococcus pneumoniae.

A major Streptococcus pneumoniae pathogenic factor is the cholesterol-dependent cytolysin pneumolysin, binding membrane cholesterol and producing permanent lytic or transient pores. During brain infections, vascular damage with variable ischemia occurs. The role of ischemia on pneumolysin's pore-forming capacity remains unknown. In acute brain slice cultures and primary cultured glia, we studied acute toxin lysis (via propidium iodide staining and LDH release) and transient pore formation (by analyzing increases in the intracellular calcium). We analyzed normal peripheral tissue glucose conditions (80 mg%), normal brain glucose levels (20 mg%), and brain hypoglycemic conditions (3 mg%), in combinations either with normoxia (8% oxygen) or hypoxia (2% oxygen). At 80 mg% glucose, hypoxia enhanced cytolysis via pneumolysin. At 20 mg% glucose, hypoxia did not affect cell lysis, but impaired calcium restoration after non-lytic pore formation. Only at 3 mg% glucose, during normoxia, did pneumolysin produce stronger lysis. In hypoglycemic (3 mg% glucose) conditions, pneumolysin caused a milder calcium increase, but restoration was missing. Microglia bound more pneumolysin than astrocytes and demonstrated generally stronger calcium elevation. Thus, our work demonstrated that the toxin pore-forming capacity in cells continuously diminishes when oxygen is reduced, overlapping with a continuously reduced ability of cells to maintain homeostasis of the calcium influx once oxygen and glucose are reduced.

Acute Rift Valley fever virus infection induces inflammatory cytokines and cell death in ex vivo rat brain slice culture.

Rift Valley fever virus (RVFV) is an emerging arboviral disease with pandemic potential. While infection is often self-limiting, a subset of individuals may develop late-onset encephalitis, accounting for up to 20 % of severe cases. Importantly, individuals displaying neurologic disease have up to a 53 % case fatality rate, yet the neuropathogenesis of RVFV infection remains understudied. In this study, we evaluated whether ex vivo postnatal rat brain slice cultures (BSCs) could be used to evaluate RVFV infection in the central nervous system. BSCs mounted an inflammatory response after slicing, which resolved over time, and they were viable in culture for at least 12 days. Infection of rat BSCs with pathogenic RVFV strain ZH501 induced tissue damage and apoptosis over 48 h. Viral replication in BSCs reached up to 1×107 p.f.u. equivalents/ml, depending on inoculation dose. Confocal immunofluorescent microscopy of cleared slices confirmed direct infection of neurons as well as activation of microglia and astrocytes. Further, RVFV-infected rat BSCs produced antiviral cytokines and chemokines, including MCP-1 and GRO/KC. This study demonstrates that rat BSCs support replication of RVFV for ex vivo studies of neuropathogenesis. This allows for continued and complementary investigation into RVFV infection in an ex vivo postnatal brain slice culture format.

NMDA glutamate receptor antagonist MK-801 induces proteome changes in adult human brain slices which are partially counteracted by haloperidol and clozapine.

Deciphering the molecular pathways associated with N-methyl-D-aspartate receptor (NMDAr) hypofunction and its interaction with antipsychotics is necessary to advance our understanding of the basis of schizophrenia, as well as our capacity to treat this disease. In this regard, the development of human brain-derived models that are amenable to studying the neurobiology of schizophrenia may contribute to filling the gaps left by the widely employed animal models. Here, we assessed the proteomic changes induced by the NMDA glutamate receptor antagonist MK-801 on human brain slice cultures obtained from adult donors submitted to respective neurosurgery. Initially, we demonstrated that MK-801 diminishes NMDA glutamate receptor signaling in human brain slices in culture. Next, using mass-spectrometry-based proteomics and systems biology in silico analyses, we found that MK-801 led to alterations in proteins related to several pathways previously associated with schizophrenia pathophysiology, including ephrin, opioid, melatonin, sirtuin signaling, interleukin 8, endocannabinoid, and synaptic vesicle cycle. We also evaluated the impact of both typical and atypical antipsychotics on MK-801-induced proteome changes. Interestingly, the atypical antipsychotic clozapine showed a more significant capacity to counteract the protein alterations induced by NMDAr hypofunction than haloperidol. Finally, using our dataset, we identified potential modulators of the MK-801-induced proteome changes, which may be considered promising targets to treat NMDAr hypofunction in schizophrenia. This dataset is publicly available and may be helpful in further studies aimed at evaluating the effects of MK-801 and antipsychotics in the human brain.

Herpes simplex virus infection induces necroptosis of neurons and astrocytes in human fetal organotypic brain slice cultures.

Herpes simplex virus (HSV) encephalitis (HSE) is a serious and potentially life-threatening disease, affecting both adults and newborns. Progress in understanding the virus and host factors involved in neonatal HSE has been hampered by the limitations of current brain models that do not fully recapitulate the tissue structure and cell composition of the developing human brain in health and disease. Here, we developed a human fetal organotypic brain slice culture (hfOBSC) model and determined its value in mimicking the HSE neuropathology in vitro. Cell viability and tissues integrity were determined by lactate dehydrogenase release in supernatant and immunohistological (IHC) analyses. Brain slices were infected with green fluorescent protein (GFP-) expressing HSV-1 and HSV-2. Virus replication and spread were determined by confocal microscopy, PCR and virus culture. Expression of pro-inflammatory cytokines and chemokines were detected by PCR. Cell tropism and HSV-induced neuropathology were determined by IHC analysis. Finally, the in situ data of HSV-infected hfOBSC were compared to the neuropathology detected in human HSE brain sections. Slicing and serum-free culture conditions were optimized to maintain the viability and tissue architecture of ex vivo human fetal brain slices for at least 14days at 37°C in a CO2 incubator. The hfOBSC supported productive HSV-1 and HSV-2 infection, involving predominantly infection of neurons and astrocytes, leading to expression of pro-inflammatory cytokines and chemokines. Both viruses induced programmed cell death-especially necroptosis-in infected brain slices at later time points after infection. The virus spread, cell tropism and role of programmed cell death in HSV-induced cell death resembled the neuropathology of HSE. We developed a novel human brain culture model in which the viability of the major brain-resident cells-including neurons, microglia, astrocytes and oligodendrocytes-and the tissue architecture is maintained for at least 2weeks in vitro under serum-free culture conditions. The close resemblance of cell tropism, spread and neurovirulence of HSV-1 and HSV-2 in the hfOBSC model with the neuropathological features of human HSE cases underscores its potential to detail the pathophysiology of other neurotropic viruses and as preclinical model to test novel therapeutic interventions.

Preclinical translational platform of neuroinflammatory disease biology relevant to neurodegenerative disease

Neuroinflammation is a key driver of neurodegenerative disease, however the tools available to model this disease biology at the systems level are lacking. We describe a translational drug discovery platform based on organotypic culture of murine cortical brain slices that recapitulate disease-relevant neuroinflammatory biology. After an acute injury response, the brain slices assume a chronic neuroinflammatory state marked by transcriptomic profiles indicative of activation of microglia and astrocytes and loss of neuronal function. Microglia are necessary for manifestation of this neuroinflammation, as depletion of microglia prior to isolation of the brain slices prevents both activation of astrocytes and robust loss of synaptic function genes. The transcriptomic pattern of neuroinflammation in the mouse platform is present in published datasets derived from patients with amyotrophic lateral sclerosis, Huntington’s disease, and frontotemporal dementia. Pharmacological utility of the platform was validated by demonstrating reversal of microglial activation and the overall transcriptomic signature with transforming growth factor-β. Additional anti-inflammatory targets were screened and inhibitors of glucocorticoid receptors, COX-2, dihydrofolate reductase, and NLRP3 inflammasome all failed to reverse the neuroinflammatory signature. Bioinformatics analysis of the neuroinflammatory signature identified protein tyrosine phosphatase non-receptor type 11 (PTPN11/SHP2) as a potential target. Three structurally distinct inhibitors of PTPN11 (RMC-4550, TN0155, IACS-13909) reversed the neuroinflammatory disease signature. Collectively, these results highlight the utility of this novel neuroinflammatory platform for facilitating identification and validation of targets for neuroinflammatory neurodegenerative disease drug discovery.

Human organotypic brain slice cultures: a detailed and improved protocol for preparation and long-term maintenance

The investigation of the human brain at cellular and microcircuit level remains challenging due to the fragile viability of neuronal tissue, inter- and intra-variability of the samples and limited availability of human brain material. Especially brain slices have proven to be an excellent source to investigate brain physiology and disease at cellular and small network level, overcoming the temporal limits of acute slices. Here we provide a revised, detailed protocol of the production and in-depth knowledge on long-term culturing of such human organotypic brain slice cultures for research purposes. We highlight the critical pitfalls of the culturing process of the human brain tissue and present exemplary results on viral expression, single-cell Patch-Clamp recordings, as well as multi-electrode array recordings as readouts for culture viability, enabling the use of organotypic brain slice cultures of these valuable tissue samples for basic neuroscience and disease modeling (Fig. 1).

P-tau Ser356 is associated with Alzheimer’s disease pathology and is lowered in brain slice cultures using the NUAK inhibitor WZ4003

Tau hyperphosphorylation and aggregation is a common feature of many dementia-causing neurodegenerative diseases. Tau can be phosphorylated at up to 85 different sites, and there is increasing interest in whether tau phosphorylation at specific epitopes, by specific kinases, plays an important role in disease progression. The AMP-activated protein kinase (AMPK)-related enzyme NUAK1 has been identified as a potential mediator of tau pathology, whereby NUAK1-mediated phosphorylation of tau at Ser356 prevents the degradation of tau by the proteasome, further exacerbating tau hyperphosphorylation and accumulation. This study provides a detailed characterisation of the association of p-tau Ser356 with progression of Alzheimer’s disease pathology, identifying a Braak stage-dependent increase in p-tau Ser356 protein levels and an almost ubiquitous presence in neurofibrillary tangles. We also demonstrate, using sub-diffraction-limit resolution array tomography imaging, that p-tau Ser356 co-localises with synapses in AD postmortem brain tissue, increasing evidence that this form of tau may play important roles in AD progression. To assess the potential impacts of pharmacological NUAK inhibition in an ex vivo system that retains multiple cell types and brain-relevant neuronal architecture, we treated postnatal mouse organotypic brain slice cultures from wildtype or APP/PS1 littermates with the commercially available NUAK1/2 inhibitor WZ4003. Whilst there were no genotype-specific effects, we found that WZ4003 results in a culture-phase-dependent loss of total tau and p-tau Ser356, which corresponds with a reduction in neuronal and synaptic proteins. By contrast, application of WZ4003 to live human brain slice cultures results in a specific lowering of p-tau Ser356, alongside increased neuronal tubulin protein. This work identifies differential responses of postnatal mouse organotypic brain slice cultures and adult human brain slice cultures to NUAK1 inhibition that will be important to consider in future work developing tau-targeting therapeutics for human disease.

Effects of corticosterone and amyloid beta on the corticosteroid receptors in organotypic brain slice cultures

Effects of corticosterone and amyloid beta on the corticosteroid receptors in organotypic brain slice cultures

Early Age- and Sex-Dependent Regulation of Astrocyte-Mediated Glutamatergic Synapse Elimination in the Rat Prefrontal Cortex: Establishing an Organotypic Brain Slice Culture Investigating Tool.

Clinical and pre-clinical studies of neuropsychiatric (NP) disorders show altered astrocyte properties and synaptic networks. These are refined during early postnatal developmental (PND) stages. Thus, investigating early brain maturational trajectories is essential to understand NP disorders. However, animal experiments are highly time-/resource-consuming, thereby calling for alternative methodological approaches. The function of MEGF10 in astrocyte-mediated synapse elimination (pruning) is crucial to refine neuronal networks during development and adulthood. To investigate the impact of MEGF10 during PND in the rat prefrontal cortex (PFC) and its putative role in brain disorders, we established and validated an organotypic brain slice culture (OBSC) system. Using Western blot, we characterized the expression of MEGF10 and the synaptic markers synaptophysin and PSD95 in the cortex of developing pups. We then combined immunofluorescent-immunohistochemistry with Imaris-supported 3D analysis to compare age- and sex-dependent astrocyte-mediated pruning within the PFC in pups and OBSCs. We thereby validated this system to investigate age-dependent astrocyte-mediated changes in pruning during PND. However, further optimizations are required to use OBSCs for revealing sex-dependent differences. In conclusion, OBSCs offer a valid alternative to study physiological astrocyte-mediated synaptic remodeling during PND and might be exploited to investigate the pathomechanisms of brain disorders with aberrant synaptic development.

Evaluating dynamic autophagic responses to tau inclusions

AbstractBackgroundAlzheimer’s disease and other tauopathies are characterised by the accumulation of misfolded microtubule‐associated protein tau in intracellular inclusions. We recently showed that newly formed tau inclusions are dynamic structures with a slower, but constant turnover of aggregated tau in murine organotypic brain slice cultures (BSCs) (Croft et al., 2021). Clearance by autophagy or the proteasomal machinery are indicated to be the key mechanisms involved in pathogenic protein clearance. However, the current tools to measure these processes have received mixed review, and in particular struggle to capture these processes in a continuous manner.MethodsTo dynamically assess autophagy in the presence of tau inclusions we have repurposed the autophagic flux sensor (GFP‐LC3‐RFP∆G‐LC3) to be delivered by rAAV to specific central nervous system (CNS) cell types in BSCs. This probe delivers a cytosolic internal control so autophagic flux can be estimated by calculating the GFP/RFP ratio. When utilized in conjunction with live imaging, the rate of autophagic flux can be measured over time in the same CNS cells expressing pathological or physiological tau. The rate of autophagic flux can also be measured in response to established/novel compounds or genetic manipulation.ResultsWe have successfully developed and characterized the use of rAAV‐GFP‐LC3‐RFP∆G‐LC3 specifically in neurons and astrocytes in BSCs and are expanding its use to other CNS cell types. We have validated its use to sense autophagic flux whilst pharmacologically enhancing and inhibiting autophagy in BSCs. We are now using this sensor to determine dynamic autophagic processes in the presence of soluble tau and aggregated tau and its response to other manipulations.ConclusionsMethods to understand autophagic processes have traditionally only provided snapshots over time. With this tool we can now dynamically assess autophagy in ex vivo BSCs and characterize changes in response to tau and other pathologies. Overall, understanding the mechanisms that enable tau turnover may guide therapies for Alzheimer’s disease and other neurodegenerative conditions where tau plays a role. Importantly, we will also begin to understand whether enhancing tau inclusion clearance may be beneficial or detrimental to cell health.

New in vitro tools to investigate the role of microglia in neurodegeneration

AbstractBackgroundHuman induced pluripotent stem cells (iPSCs) have transpired as an attractive tool to investigate neurological diseases. And while immune cells, and in particular microglia, have emerged as important players in many neurological and in particular in neurodegenerative diseases, it remains a challenge to fully recapitulate an in vivo‐like phenotype ex vivo. This is due to the fact that microglial identity is shaped by the neural tissue environment.MethodWe have now developed a chimeric brain slice culture system, where we engraft iPSC‐derived microglia onto mouse (and human) organotypic brain slice cultures. Brain slice cultures offer the advantage that many aspects of the complex cytoarchitecture of the mature/aged living brain, including cortical layering, columnar organization and electrophysiological properties can be preserved over several weeks.ResultWe found that iPSC‐derived microglial precursors integrate and differentiate well in the brain slice cultures, with morphology, network characteristics and functional responses reminiscent of human microglia. Gene expression profiling of iPSC‐derived microglia revealed enhanced maturation over time. Upon induction of neurodegenerative disease pathology in these slice cultures, the iPSC‐derived microglia show transcriptional changes converging towards those observed in patients with neurodegenerative disease pathology.ConclusionThis system is a novel cellularly complex tool allowing to dissect the dynamics and molecular mechanisms of microglial genetic risk factors of many neurological diseases, while preserving experimental amenability to test therapeutic approaches in a human(ized) system.

Modulation of cannabinoid receptor‐2 alters brain clearance of human tau

AbstractBackgroundMultiple studies have demonstrated a strong neuroinflammatory response in the presence of Tau pathology, which is strongly associated with the clinical symptoms and cognitive decline found in Alzheimer’s disease (AD). The correlation between AD progression and tau pathologies, rather than Ab accumulation, suggests that targeting pathological tau may be a more effective therapeutic approach. Microglia have been implicated in tauopathies as they may contribute to tau phosphorylation and aggregation and the uptake and spread of seed‐competent tau to healthy neurons due to dysfunctional degradation. Cannabinoid type 2 receptors (CB2) are highly expressed in immune cells and upregulated in activated microglia under conditions of neurologic disease, such as AD. The pharmacological modulation of CB2 have demonstrated a reduction in inflammation and plaque deposition, suggesting a role of CB2 on the immune system to influence AD‐related pathologies and phenotypes. However, there are limited findings on the CB2 regulation of tau accumulation.MethodWe have generated organotypic brain slice cultures from CB2‐EGFP/f/f mice and transduced with rAAV‐P301L/S320F‐human tau, which previously has shown to accumulate total tau and develop insoluble tau species. Slices were treated with CB2‐selective compounds and evaluated for tau pathology and microglial phenotypes using real‐time imaging and biochemical analyses.ResultPrevious investigation in our lab of a synucleinopathy animal model revealed the capacity of CB2 signaling to alter the function of immune cells and reduce accumulation of phosphorylated human alpha‐synuclein. Furthermore, we have determined that treatment with a CB2 inverse agonist will increase phagocytosis of pHrodo E. coli bioparticles in microglia BV2 cultures stimulated by LPS. Our preliminary work in brain slices over‐expressing mutant human tau show a trend toward reduced ratio of phosphorylated tau (Ser202/Thr205) to total tau in the soluble protein fraction (p = 0.0549) in slices treated with CB2 inverse agonist SR144528 compared to vehicle. We are repeating the experiment and performing further evaluations in organotypic brain slice cultures from CB2‐KO mice.ConclusionThese studies will advance our understanding of the role of brain CB2 in pathologic tau removal and potentially highlight CB2 as a therapeutic target against tauopathies such as AD.

Hyper‐sensitive interferon response in a mouse model of Alzheimer’s disease‐Down syndrome

AbstractBackgroundDown syndrome (DS) is caused by trisomy of chromosome 21 (Hsa21) and leads to an elevated risk of early‐onset Alzheimer’s disease. People with DS have a perturbed immune system including an over‐activated interferon response, which is likely due to four genes encoding interferon receptors being located on Hsa21 (1). How this affects inflammation in the brain is not well understood. In this study, we investigate if an extra copy of these genes is sufficient to elevate the interferon response in the brain of mouse models of DS, using in vivo and ex vivo approaches.MethodsThe Dp1Tyb (MGI:5703798) mouse has three‐copies of ∼148 Hsa21 orthologous genes, while the Dp2Tyb (MGI:5703800) mouse has a sub‐region of ∼32 of those Hsa21 orthologous genes in three‐copies, including the four interferon receptor genes. Organotypic brain slice cultures (OBSCs) were prepared from P7‐P9 pups and treated with interferon‐β (24 hours). Slices were collected for western blotting, qPCR, or immunohistochemistry, and media for CXCL10 ELISA.Dp2Tyb mice were crossed with AppNL‐G‐F (MGI:5637817) mice to generate four genotypes, to determine if the amyloid‐β interferon response is elevated by the additional copy of the Dp2Tyb region. Mice were tested in the light‐dark box and marble burying test to assay the effect on anxiety. Amyloid accumulation and interferon response in the brain will be determined by immunofluorescence, MSD amyloid‐β assay and qPCR.ResultsBoth Dp1Tyb and Dp2Tyb OBSCs had a hypersensitive response to interferon‐β treatment, with higher expression of interferon‐stimulated genes in lysed slices and higher levels of CXCL10 protein secreted into the media.ConclusionBoth DS preclinical models displayed a hypersensitive interferon response, indicating that interferon hypersensitivity may occur in the brain of individuals with DS and is likely due to having three‐copies of the interferon receptor genes encoded on Hsa21. Amyloid pathology has been shown to induce an interferon response (2,3). How interferon hypersensitivity in individuals with Down syndrome affects the neuroinflammatory response to amyloid pathology is unknown. To investigate this, we have crossed the Dp2Tyb model with the AppNL‐G‐F mouse model of amyloid pathology.

TMIC-61. INVESTIGATING REPROGRAMING OF THE GBM’S TUMOR MICROENVIRONMENT AS A RESULT OF CO-TRANSFER OF HEMATOPOIETIC STEM CELLS (HSCS) WITH ADOPTIVE CELLULAR THERAPY

Abstract Glioblastoma (GBM) is an incredibly aggressive and prevalent primary CNS tumor with dismal survival outcomes. GBM’s intra-tumor heterogeneity and the powerful regulatory mechanisms within the tumor microenvironment (TME) that hamper immune activation have proved to be formidable challenges to the development of effective therapies. Fortunately, immunotherapy has emerged as a potentially powerful approach to achieve of immune activation within the TME through adoptive immunotherapy and checkpoint inhibition Our previous work has demonstrated that the survival benefit of adoptive cellular therapy (ACT) is significantly enhanced by concomitant transfer of bone marrow derived hematopoietic stem cells (HSC) with tumor-reactive T cells in preclinical models. Using a novel organotypic brain slice culture (BSC) model study using glioma-bearing C57BL/6J mice, we interrogate tumor microenvironment and directly demonstrate that the presence of HSC-derived cells in the microenvironment increases T cell migration to tumor, and increased tumor cell death. In addition, we can investigate reprogramming of the TME as a result of treatment with this ACT+HSC platform. These studies are unique in that through this model system, we are capable of studying the cell-cell interactions occurring within the tumor microenvironment TME longitudinally while accounting for the complexity and the spatial orientation of the components within the TME that promote immunosuppression and tumor escape.

MODL-44. DEVELOPMENT OF A NOVEL, PERSONALIZED DRUG SENSITIVITY SCORE TO ASSESS TUMOR KILLING AND TOXICITY WITHIN AN ORGANOTYPIC BRAIN SLICE CULTURE PLATFORM

Abstract Development of new therapeutics for clinical use requires balancing tumor killing and off-target toxicity to normal tissue. We have established an organotypic brain slice culture (OBSC) platform which enables us to assess both on- and off-target drug effects via engraftment, treatment, and analysis of cell lines and uncultured patient brain tumor tissue. We have used OBSCs to test seven cell lines, ten patient tissues, and normal brain tissue against a panel of eleven different therapeutics. To determine a normalized comparison among drugs of differing potencies, we have established a drug sensitivity score (DSS) which combines both direct tumor killing via bioluminescence imaging and off-target toxicity via propidium iodide staining. Our ability to define these values independently allows us to generate normalized therapeutic window ratios for each drug-tumor-OBSC interaction. From these results, 11 different parameters, including those commonly used to define drug activity such as IC10, IC50, and area under the curve, were assessed and combined into a single ranked score which combines more than 100 data points. This algorithm enables relative comparisons among tumor responses to several drugs with less bias toward more potent compounds which produce correspondingly high off-target toxicity. Trends across DSSs for every tumor indicate that we can calculate scores throughout the entire scoring range. While we find that patient tumor tissue is generally more sensitive to treatment than cell lines, there is still a wide distribution of patient tumor sensitivities among tumor types and therapeutics. Additionally, our DSS can detect differences in tumor sensitivity after a single gene alteration in RAD18. In summary, our DSS algorithm and scoring system provides a summarized, normalized way to assess both on-target killing and off-target toxicity, allowing more comprehensive comparisons of drugs with varying potency.

CNSC-10. GLIOBLASTOMA CELLS IMITATE NEURON’S EXCITABILITY IN ACUTE AND ORGANOTYPIC HUMAN BRAIN SLICES

Abstract Glioblastoma (GBM) is a highly lethal cancer due to its inter- and intra-patient molecular and physiological heterogeneity, and its complex interaction with brain tissue. Recent findings demonstrate that Neuron-to-brain tumor-synaptic-communications (NBTSCs) engage in a vicious cycle of enhanced neuronal activity, driving glioma progression and invasion and tumor-induced epilepsy. However, the physiology of these synaptic interactions in the tumor-infiltrated human neocortex remains largely uncharacterized. To address this gap, our study focused on investigating the intrinsic electrical properties of glioma cells and NBTSCs in the tumor-infiltrated human cortex using a unique experimental workflow involving acute and cultured human tumor-infiltrated brain slices. Via an adeno-associated virus-assisted gene delivery technique, we selectively express fluorescence proteins in a cell-type specific manner. By performing fluorescence-guided whole-cell patch clamp recording, we characterized the electrophysiological properties of glioma cells and neurons under the tumor microenvironment. We observed that glioma cells from tumor-infiltrated human brain samples displayed depolarized resting membrane potential (-32.21 ± 2.1mV) and high input resistance (1.24 ± 0.18 GΩ). Approximately 69.2% of glioma cells exhibited spikelets or distorted-action potentials upon current injection. Consistent with other studies, a subset of glioma cells (11.8%) exhibited spontaneous excitatory postsynaptic currents (sEPSC). In addition, the tumor-infiltrated microenvironment significantly influences the proximal neurons’ physiology and the neuronal microcircuitry, leading to abnormal intrinsic electrical properties, depolarized resting membrane potential (-37.66 ± 4.32 mV), and a significant reduction in spontaneous synaptic input frequency. Collectively, our study consolidates the existence of NBTSCs in human cancer patients and reveals that glioma cells exhibit neuron-like excitability, which is contrary to the common understanding that they are considered as non-excitable. Under the tumor microenvironment, neurons display hyperexcitability. Our newly established organotypic human brain slice culture platform provides potential grounds for exploring further the biological characteristics of gliomas and NBTSCs.

CNSC-39. GENERATION AND CHARACTERIZATION OF LIVING ORGANOTYPIC BRAIN SLICE CULTURES AS A NOVEL PRE-CLINICAL MODEL FOR BRAIN CANCER

Abstract A challenge in cancer research is developing reproducible, reliable, and practical models which can capture the complexity of cancer development and treatment. The development of functional precision medicine platforms is emerging as a promising strategy for improving pre-clinical drug testing and guiding clinical decisions. We have developed an organotypic brain slice culture (OBSC) technology composed of intact multicellular tissue which can be rapidly used for spatiotemporal drug response testing. OBSCs are reproducibly generated from Sprague-Dawley rat pups and used as living tissue substrates to culture treat different tumor cell lines and uncultured patient brain tumor resection tissue. We evaluated OBSC quality and reproducibility throughout the study by using a propidium iodide nuclear permeability assay. This technique enabled a broad dynamic range to distinguish between healthy and unhealthy slices. In these studies, the rat pup age at the time of generation had an impact on OBSC quality and quantity, indicating that OBSCs should be generated from eight-day-old pups. We also found that optimal OBSCs were generated using improved methods for brain dissection and OBSC culture conditions, establishing our robust, standardized procedure for OBSC generation. Following this optimization period, we continued to conduct quality control analysis with 6 OBSCs per batch for reproducibility. We also conducted immunohistochemistry analysis immediately after slicing and concluded that morphology of neurons in OBSCs remained unchanged between day 0 and 4. In addition, the activation of astrocytes attenuates by day 4 but persists in macrophages/microglia, suggesting that the myeloid cells can phagocytose dead cells and debris in OBSCs. In summary, these results indicate that the OBSC platform may be an effective model that accelerates preclinical drug testing and directs drug development towards clinical evaluation.

MODL-28. LIVING EX VIVO ORGANOTYPIC BRAIN SLICE CULTURES AS A COMPREHENSIVE SUBSTRATE FOR TUMOR ENGRAFTMENT AND ANALYSIS

Abstract Heterogeneity, drug resistance, tumor infiltration, and invasiveness significantly contribute to the poor prognosis often associated with brain cancers. To enhance drug development, it is crucial to have models that accurately replicate human responses. Standard in vitro methods to culture, treat and analyze brain tumors often lack resemblance to the original tumor and provide limited insight. To address these challenges, we developed an organotypic brain slice culture (OBSC) model that recapitulates in vivo tumor characteristics within a functional tumor microenvironment (TME) and provides rapid results. OBSCs effectively measure engrafted tumor growth, morphology, and invasion rates. Furthermore, engrafted tumors can undergo relevant therapeutic treatments and be analyzed for drug outcomes. In a 4-day assay, we successfully engrafted a diverse range of brain cancer cell lines, including low-passage lines that struggle to establish in vivo. These cell lines maintain tumor-specific invasion patterns, interact with the OBSC microenvironment, and display killing patterns consistent with in vivo tumor responses. Notably, invasive, metastatic, and diffuse tumor cell lines migrate outward on OBSCs, while densely growing lines contract inward. To further validate OBSCs as a model for the TME, we treated MB231BR, LN229, and U373 tumor lines with TR107, an experimental ClpP inhibitor derived from ONC201. This therapeutic family maximizes tumor elimination by targeting normal cells within the TME. In vitro experiments showed TR107 achieving incomplete tumor kill on cell lines grown in vitro, whereas treatment on OBSCs led to complete tumor kill, suggesting OBSCs' contribution to TME-based tumor elimination. Conclusively, OBSCs provide a valuable platform for studying brain cancers and accurately replicating tumor behavior and responses to therapies within the TME. This model has the potential to advance drug development and deepen our understanding of brain cancer biology, ultimately improving prognoses and treatment outcomes for patients.

MODL-48. AUTOMATING ANALYSIS OF ORGANOTYPIC BRAIN SLICE CULTURE USING MACHINE LEARNING-POWERED COMPUTER VISION

Abstract The difficulty of image labeling and analysis poses a problem for many areas of scientific research, including the recently developed Organotypic Brain Slice Culture (OBSC) tumor characterization platform. An OBSC experiment generates dozens of images which, up to this point, had to be manually quantified. In order to increase the efficiency, consistency, and accuracy of the OBSC image analysis process, we have created a computer program which automates the analysis of OBSC images. This platform uses a number of computer vision (CV) techniques to identify objects of interest and measure their corresponding signals. For fluorescent images of single tumor spots, a straightforward thresholding algorithm divides the image into foreground and background. This automated approach substantially reduces analysis time, and the thresholding algorithm measures unusually shaped tumor spots up to 9% more accurately than previously used manual methods. Other images capture an entire plate of up to 12 OBSCs, complicating the image analysis task. A machine-learning algorithm trained on the web-based platform Biodock could identify individual OBSCs, even if they were touching each other or the sides of the wells. The ML-powered methods consistently reproduced killing curves for treatment effects on tumor tissue as well as the brain tissue substrate, and these results were obtained in much less time. By combining various CV approaches to image analysis, we have completely automated the image analysis component of our OBSC platform. This approach could be adapted to other tumor co-culture platforms.

MODL-03. AN EX VIVO PLATFORM BASED ON ORGANOTYPIC BRAIN SLICE CULTURE FOR INFORMING PERSONALIZED BRAIN CANCER THERAPY

Abstract While the improved classification of CNS tumors is already enhancing the scientific rigor of clinical brain cancer treatment guidelines, the impact of these integrated diagnoses based on histopathological features or molecular insight on precision oncology medicine has yet to demonstrate widespread clinical benefit. Patient tumor-derived cancer models, including patient tumor-derived cell lines, organoids, and explants, have provided practical models based on a patient’s individual tumor for effective therapeutic drugs. Importantly, patient tumor-derived organoids have shown potential to guide personalized care and optimize clinical outcomes by functionally predicting patient response to antitumor drugs prior to in vivo treatment. Our team has built an organotypic brain slice culture (OBSC)-based platform and multi-parametric algorithm that allows for the ex vivo engraftment, rapid treatment, and systemic analysis of resected patient brain tumor tissue. This OBSC platform has successfully supported engraftment of each patient tumor tested to this point, including high- and low-grade adult and pediatric tumor tissue. The established patient tumor-derived organoids show the ability to activate the endogenous astrocytes within OBSCs, which have been reported in brain tumor. Whole exome sequencing analysis showed that patient tumor tissue engrafted onto OBSCs maintained a significant genetic resemblance to their parent tumor, while tumor tissue expanded in vitro displayed a distinctly different DNA profile. Additionally, our algorithm calculates dose-response relationships of both tumor kill and normal brain tissue toxicity, generating summarized drug sensitivity scores based on therapeutic window and allowing us to normalize response profiles across a panel of FDA-approved and exploratory agents. Summarized patient tumor scores after OBSC treatment revealed positive associations to clinical outcomes, suggesting the OBSC platform can provide rapid, accurate, functional testing to ultimately guide patient care.