Cell Types Research Articles

Deciphering spatial domains from spatial multi-omics with SpatialGlue.

Advances in spatial omics technologies now allow multiple types of data to be acquired from the same tissue slice. To realize the full potential of such data, we need spatially informed methods for data integration. Here, we introduce SpatialGlue, a graph neural network model with a dual-attention mechanism that deciphers spatial domains by intra-omics integration of spatial location and omics measurement followed by cross-omics integration. We demonstrated SpatialGlue on data acquired from different tissue types using different technologies, including spatial epigenome-transcriptome and transcriptome-proteome modalities. Compared to other methods, SpatialGlue captured more anatomical details and more accurately resolved spatial domains such as the cortex layers of the brain. Our method also identified cell types like spleen macrophage subsets located at three different zones that were not available in the original data annotations. SpatialGlue scales well with data size and can be used to integrate three modalities. Our spatial multi-omics analysis tool combines the information from complementary omics modalities to obtain a holistic view of cellular and tissue properties.

Increasing adult neurogenesis protects mice from epilepsy.

Neurogenesis occurs in the adult brain in the hippocampal dentate gyrus, an area that contains neurons which are vulnerable to insults and injury, such as severe seizures. Previous studies showed that increasing adult neurogenesis reduced neuronal damage after these seizures. Because the damage typically is followed by chronic life-long seizures (epilepsy), we asked if increasing adult neurogenesis would prevent epilepsy. Adult neurogenesis was selectively increased by deleting the pro-apoptotic gene Bax from Nestin-expressing progenitors. Tamoxifen was administered at 6 weeks of age to conditionally delete Bax in Nestin-CreER T2 Bax fl/fl mice. Six weeks after tamoxifen administration, severe seizures (status epilepticus; SE) were induced by injection of the convulsant pilocarpine. After mice developed epilepsy, seizure frequency was quantified for 3 weeks. Mice with increased adult neurogenesis exhibited fewer chronic seizures. Postictal depression was reduced also. These results were primarily in female mice, possibly because they were the more affected by Bax deletion than males, consistent with sex differences in Bax . The female mice with enhanced adult neurogenesis also showed less neuronal loss of hilar mossy cells and hilar somatostatin-expressing neurons than wild type females or males, which is notable because these two hilar cell types are implicated in epileptogenesis. The results suggest that selective Bax deletion to increase adult neurogenesis can reduce experimental epilepsy, and the effect shows a striking sex difference. The results are surprising in light of past studies showing that suppressing adult-born neurons can also reduce chronic seizures.

Automated single-cell omics end-to-end framework with data-driven batch inference.

To facilitate single-cell multi-omics analysis and improve reproducibility, we present SPEEDI (Single-cell Pipeline for End to End Data Integration), a fully automated end-to-end framework for batch inference, data integration, and cell type labeling. SPEEDI introduces data-driven batch inference and transforms the often heterogeneous data matrices obtained from different samples into a uniformly annotated and integrated dataset. Without requiring user input, it automatically selects parameters and executes pre-processing, sample integration, and cell type mapping. It can also perform downstream analyses of differential signals between treatment conditions and gene functional modules. SPEEDI's data-driven batch inference method works with widely used integration and cell-typing tools. By developing data-driven batch inference, providing full end-to-end automation, and eliminating parameter selection, SPEEDI improves reproducibility and lowers the barrier to obtaining biological insight from these valuable single-cell datasets. The SPEEDI interactive web application can be accessed at https://speedi.princeton.edu/ .

Probing the structure of water in individual living cells

Water regulates or even governs a wide range of biological processes. Despite its fundamental importance, surprisingly little is known about the structure of intracellular water. Herein we employ a Raman micro-spectroscopy technique to uncover the composition, abundance and vibrational spectra of intracellular water in individual living cells. In three different cell types, we show a small but consistent population (~3%) of non-bulk-like water. It exhibits a weakened hydrogen-bonded network and a more disordered tetrahedral structure. We attribute this population to biointerfacial water located in the vicinity of biomolecules. Moreover, our whole-cell modeling suggests that all soluble (globular) proteins inside cells are surrounded by, on average, one full molecular layer (about 2.6 Angstrom) of biointerfacial water. Furthermore, relative invariance of biointerfacial water is observed among different single cells. Overall, our study not only opens up experimental possibilities of interrogating water structure in vivo but also provides insights into water in life.

Triphenylphosphonium-Conjugated Palmitic Acid for Mitochondrial Targeting of Pancreatic Cancer Cells: Proteomic and Molecular Evidence.

Pancreatic ductal adenocarcinoma (PDAC)'s resistance to therapies is mainly attributed to pancreatic cancer stem cells (PCSCs). Mitochondria-impairing agents can be used to hamper PCSC propagation and reduce PDAC progression. Therefore, to develop an efficient vector for delivering drugs to the mitochondria, we synthesized tris(3,5-dimethylphenyl)phosphonium-conjugated palmitic acid. Triphenylphosphonium (TPP) is a lipophilic cationic moiety that promotes the accumulation of conjugated agents in the mitochondrion. Palmitic acid (PA), the most common saturated fatty acid, has pro-apoptotic activity in different types of cancer cells. TPP-PA was prepared by the reaction of 16-bromopalmitic acid with TPP, and its structure was characterized by 1H and 13C NMR and HRMS. We compared the proteomes of TPP-PA-treated and untreated PDAC cells and PCSCs, identifying dysregulated proteins and pathways. Furthermore, assessments of mitochondrial membrane potential, intracellular ROS, cardiolipin content and lipid peroxidation, ER stress, and autophagy markers provided information on the mechanism of action of TPP-PA. The findings showed that TPP-PA reduces PDAC cell proliferation through mitochondrial disruption that leads to increased ROS, activation of ER stress, and autophagy. Hence, TPP-PA might offer a new approach for eliminating both the primary population of cancer cells and PCSCs, which highlights the promise of TPP-derived compounds as anticancer agents for PDAC.

NR0B2 re-educates myeloid immune cells to reduce regulatory T cell expansion and progression of breast and other solid tumors

Although survival from breast cancer has dramatically increased, many will develop recurrent, metastatic disease. Unfortunately, survival for this stage of disease remains very low. Activating the immune system has incredible promise since it has the potential to be curative. However, immune checkpoint blockade (ICB) which works through T cells has been largely disappointing for metastatic breast cancer. One reason for this is a suppressive myeloid immune compartment that is unaffected by ICB. Cholesterol metabolism and proteins involved in cholesterol homeostasis play important regulatory roles in myeloid cells. Here, we demonstrate that NR0B2, a nuclear receptor involved in negative feedback of cholesterol metabolism, works in several myeloid cell types to impair subsequent expansion of regulatory T cells (Tregs); Tregs being a subset known to be highly immune suppressive and associated with poor therapeutic response. Within myeloid cells, NR0B2 serves to decrease many aspects of the inflammasome, ultimately resulting in decreased IL1β; IL1β driving Treg expansion. Importantly, mice lacking NR0B2 exhibit accelerated tumor growth. Thus, NR0B2 represents an important node in myeloid cells dictating ensuing Treg expansion and tumor growth, thereby representing a novel therapeutic target to re-educate these cells, having impact across different solid tumor types. Indeed, a paper co-published in this issue demonstrates the therapeutic utility of targeting NR0B2.

Genetic predisposition to porto-sinusoidal vascular disorder.

Porto-sinusoidal vascular disorder is a rare liver disease. The pathophysiological mechanisms underlying the development of porto-sinusoidal vascular disorder are unknown. Isolated cases of porto-sinusoidal vascular disorder associated with gene mutations have been reported, but no overview is available. Therefore, we performed an extensive literature search to provide a comprehensive overview of gene mutations associated with porto-sinusoidal vascular disorder. We identified 34 genes and one chromosomal abnormality associated with porto-sinusoidal vascular disorder in the literature, and we describe here one additional gene mutation (TBL1XR1 mutation, leading to Pierpont syndrome). These gene mutations are associated either with extrahepatic organ involvement as part of syndromes (Adams Oliver, telomere biology disorders, retinal vasculopathy with cerebral leukoencephalopathy and systemic manifestations, immune deficiencies, cystic fibrosis, cystinosis, Williams Beuren, Turner, Pierpont) or with isolated porto-sinusoidal vascular disorder (KCNN3, DGUOK, FOPV, GIMAP5, FCHSD1, TRMT5, HRG gene mutations). Most of the cases were revealed by signs or complications of portal hypertension. When analysing the cell types in which these genes are expressed, we found that these genes are predominantly expressed in immune cells, suggesting that these cells may play a more important role in the development of porto-sinusoidal vascular disorder than previously thought. In addition, pathway analyses suggested that there may be 2 types of porto-sinusoidal vascular disorder associated with gene mutations: those resulting directly from morphogenetic abnormalities and those secondary to immune changes.

Leveraging Integrated RNA Sequencing to Decipher Adrenomedullin's Protective Mechanisms in Experimental Bronchopulmonary Dysplasia.

Bronchopulmonary dysplasia (BPD) is a chronic lung disease commonly affecting premature infants, with limited therapeutic options and increased long-term consequences. Adrenomedullin (Adm), a proangiogenic peptide hormone, has been found to protect rodents against experimental BPD. This study aims to elucidate the molecular and cellular mechanisms through which Adm influences BPD pathogenesis using a lipopolysaccharide (LPS)-induced model of experimental BPD in mice. Bulk RNA sequencing of Adm-sufficient (wild-type or Adm+/+) and Adm-haplodeficient (Adm+/-) mice lungs, integrated with single-cell RNA sequencing data, revealed distinct gene expression patterns and cell type alterations associated with Adm deficiency and LPS exposure. Notably, computational integration with cell atlas data revealed that Adm-haplodeficient mouse lungs exhibited gene expression signatures characteristic of increased inflammation, natural killer (NK) cell frequency, and decreased endothelial cell and type II pneumocyte frequency. Furthermore, in silico human BPD patient data analysis supported our cell type frequency finding, highlighting elevated NK cells in BPD infants. These results underscore the protective role of Adm in experimental BPD and emphasize that it is a potential therapeutic target for BPD infants with an inflammatory phenotype.

Distinct action signals by subregions in nucleus accumbens during STOP-change performance.

Nucleus accumbens (NAc) is thought to contribute to motivated behavior by signaling the value of reward-predicting cues and the delivery of anticipated reward. NAc is subdivided into core and shell, with each region containing different populations of neurons that increase or decrease firing to rewarding events. While there are various and numerous theories of functions pertaining to these subregions and cell types, most are in the context of reward processing, with fewer considering that NAc might serve functions related to action selection more generally. We recorded from single neurons in NAc as rats of both sexes performed a STOP-change task that is commonly used to study motor control and impulsivity. In this task, rats respond quickly to a spatial cue on 80% of trials (GO) and must stop and redirect planned movement on 20% of trials (STOP). We found that the activity of reward-excited neurons signaled accurate response direction on GO, but not STOP trials, and that these neurons exhibited higher pre-cue firing after correct trials resulting in stronger firing during correct GO trials and errant STOP trials, while reward-inhibited neurons significantly represented response direction on STOP trials at the time of the instrumental response. Finally, the proportion of reward-excited to reward-inhibited neurons and the strength of pre-cue firing decreased as the electrode traversed NAc. We conclude that reward-excited cells (more common in core) promote proactive action selection, while reward-inhibited cells (more common in shell) contribute to accurate responding on STOP trials that require reactive suppression and redirection of behavior.Significant Statement The ability to appropriately adapt behavior is an important part of human cognition, and one that is disrupted by many neuropsychiatric disorders. Here we recorded from neurons in the nucleus accumbens (NAc) as rats performed a cognitive control task and found cell type- and subregion-specific firing patterns. Core and reward-excited cells track trial outcome history, proactively driving behavior to the first cue-a strategy that is appropriate for most trials. Conversely, shell and reward-inhibited neurons signal accurate response direction on trials requiring redirection of behavior. Together, these data suggest that NAc neuronal populations differentially contribute to action selection.

PhysiCell Studio: a graphical tool to make agent-based modeling more accessible.

Defining a multicellular model can be challenging. There may be hundreds of parameters that specify the attributes and behaviors of objects. In the best case, the model will be defined using some format specification - a markup language - that will provide easy model sharing (and a minimal step toward reproducibility). PhysiCell is an open-source, physics-based multicellular simulation framework with an active and growing user community. It uses XML to define a model and, traditionally, users needed to manually edit the XML to modify the model. PhysiCell Studio is a tool to make this task easier. It provides a GUI that allows editing the XML model definition, including the creation and deletion of fundamental objects: cell types and substrates in the microenvironment. It also lets users build their model by defining initial conditions and biological rules, run simulations, and view results interactively. PhysiCell Studio has evolved over multiple workshops and academic courses in recent years, which has led to many improvements. There is both a desktop and cloud version. Its design and development has benefited from an active undergraduate and graduate research program. Like PhysiCell, the Studio is open-source software and contributions from the community are encouraged.

Topological optimisation and laser additive manufacturing of force-direction-sensitive NiTi porous structures with large deformation recovery behaviour

ABSTRACT Optimising the cell type and configuration is a key approach for overcoming stress concentration and irreversible deformation in lightweight lattice structures with high strength. In this work, the topology optimisation method was utilised to design force-direction-sensitive structures, including face-centre loaded (FCL), central-edge loaded (CEL), and hybrid-loaded (HL) structures, which were manufactured by laser powder bed fusion (LPBF) using Nitinol (NiTi) shape memory alloy. Results indicated that the HL lattice exhibited the highest initial peak force of 7.6 kN with higher specific compressive strength (41.74 MPa·cm3/g) and specific energy absorption (6.82 J/g), which was benefit from the layer-by-layer deformation mechanism. Furthermore, the HL lattice also exhibited the best damping properties and energy absorption capacity, while achieving a high shape recovery ratio of 85%. This work offers insights into a design strategy for functional structures with high strength and large deformation recovery capacity.

N-cadherin facilitates trigeminal sensory neuron outgrowth and target tissue innervation.

Our findings are significant because they demonstrate how neurons derived from two distinct cell populations, neural crest and placode cells, coordinate the outgrowth of their axons in time and space to generate the trigeminal ganglion using the cell adhesion molecule N-cadherin. Notably, our results provide evidence for the existence of subpopulations of neurons within the trigeminal ganglion that differentially require N-cadherin to facilitate axon outgrowth, and hint at the possibility that trigeminal pioneer neurons are derived from placode cells while followers arise from both placode and neural crest cells. These studies provide new insight into trigeminal gangliogenesis that will likely be translatable to other cranial ganglia and vertebrate species.

Abstract PO-021: Modeling marginal zone lymphomagenesis

Abstract Introduction: Marginal zone lymphomas (MZL) are the second most prevalent subtype of indolent Non-Hodgkin Lymphomas (iNHL), yet lack a unique cytogenetic identifying abnormality. The B-cell receptor signaling pathway is activated in B-cell malignancy and mediates its activity mainly through the Phosphoinositide 3-kinase (PI3K), protein kinase B (Akt), mammalian target of rapamycin (mTOR) pathway. PI3K inhibitors have demonstrated impressive clinical activity in several indolent lymphomas including MZL, underscoring the pivotal role of the PI3K-AKT-mTOR (PAM) pathway in MZL pathogenesis. Furthermore, iNHL overexpress the antiapoptotic protein Bcl-2. Methods: We developed a genetically engineered mouse model with heterozygous global knockout alleles of both the tumor suppressor genes Phosphatase and Tensin Homolog (PTEN) and Liver Kinase B1 (LKB1), resulting in heightened PAM pathway activation across all tissues. We closely monitored these mice for tumor formation via regular physical assessments over several months. Upon tumor detection, mice were euthanized, and tumors were histologically examined. For a more precise representation of B-cell biology akin to the human disease, we employed the Cre-LoxP system to create the CD19-Cre-PTENfl/fl/LKB1fl/fl and CD19-Cre-PTENfl/fl/Eµ-Bcl2. Results: Thirty mice of global KO PTEN+/- LKB1+/- succumbed to or were euthanized due to disease progression, defined as either lymph node enlargement and/or splenomegaly. By Kaplan-Meier analysis, we saw a steady decrease in both tumor-free and overall survival after 3 months of age. Median survival time was 6 months (95% CI: 6, 8). Immunohistochemical analysis of the 51 lymph nodes revealed iNHL in 61.5% (N=32), with all of the MZL subtype. The penetrance rate was 37%. Then, we looked at the absolute B cells, and the major B subtypes in age-matched mice (N=3 per group): WT, CD19-Cre-PTENfl/fl/LKB1fl/fl, CD19-Cre-PTENfl/fl/Eµ-Bcl2. We observed the following preliminary results in the absolute B cells (23,489,812; 1,550,430; 140,476,875), Marginal zone cells (1,087,883; 452,470; 42,658,701), follicular cells (19,078,598; 203,286; 57,212,627), B1 cells (962,192; 939,386; 1,803,060), and plasma cells (211,725; 202,790; 10,531,585) respectively. Then, Kruskal-Wallis tests were used to test the main effect of these three models (WT, CD19-Cre-PTENfl/fl/LKB1fl/fl and CD19-Cre-PTENfl/fl/Bcl2) on absolute cell counts for each cell type: B cells (p=0.027), follicular (p=0.061), Marginal zone (p=0.027), B1 (p=0.43), and plasma cell (p=0.066). The test results indicate that there were statistically discernible differences in absolute cell counts among the three models for B cells and MZ. Conclusion: Marginal zone lymphoma remains an incurable lymphoma with a dearth of reliable preclinical models. Our data provides, for the first time, evidence supporting the involvement of the PAM pathway in MZL pathogenesis, laying the groundwork for further research elucidating the disease biology and rational therapeutic strategies for this incurable malignancy. Citation Format: Emily Harris, Ariel Sindel, Jian He, Roy Sabo, Alden Chesney, Guanhua Lai, Adolpho Mauro, Rebecca Martins, Jennifer Koblinski, Joelene Windle, Victor Yazbeck. Modeling marginal zone lymphomagenesis [abstract]. In: Proceedings of the Fourth AACR International Meeting on Advances in Malignant Lymphoma: Maximizing the Basic-Translational Interface for Clinical Application; 2024 Jun 19-22; Philadelphia, PA. Philadelphia (PA): AACR; Blood Cancer Discov 2024;5(3_Suppl):Abstract nr PO-021.

On the possibility of using cell technologies in surgical practice

Cell therapy is one of the promising areas of modern medicine. This field has become widespread in many medical specialties, but over the past few years, cell therapy has been attracting more and more attention from specialists in the surgical field. Special attention is paid to therapy based on the use of stem cells, which is explained by their ability to self-renew and differentiate. Due to their properties, stem cells directly act in the wound site, triggering tissue regeneration mechanisms, and some types of stem cells suppress inflammation processes. In surgical practice, the following stem cells are used to correct skin defects and treat wounds: mesenchymal stem cells, induced pluripotent stem cells and embryonic stem cells, keratinocytes and fibroblasts. Each type of cell has its own advantages and is used in different directions. Many studies indicate the prospects of using cellular technologies in the treatment of diseases and wounds of various etiologies.

The transcriptomic and spatial organization of telencephalic GABAergic neuronal types.

The telencephalon of the mammalian brain comprises multiple regions and circuit pathways that play adaptive and integrative roles in a variety of brain functions. There is a wide array of GABAergic neurons in the telencephalon; they play a multitude of circuit functions, and dysfunction of these neurons has been implicated in diverse brain disorders. In this study, we conducted a systematic and in-depth analysis of the transcriptomic and spatial organization of GABAergic neuronal types in all regions of the mouse telencephalon and their developmental origins. This was accomplished by utilizing 611,423 single-cell transcriptomes from the comprehensive and high-resolution transcriptomic and spatial cell type atlas for the adult whole mouse brain we have generated, supplemented with an additional single-cell RNA-sequencing dataset containing 99,438 high-quality single-cell transcriptomes collected from the pre- and postnatal developing mouse brain. We present a hierarchically organized adult telencephalic GABAergic neuronal cell type taxonomy of 7 classes, 52 subclasses, 284 supertypes, and 1,051 clusters, as well as a corresponding developmental taxonomy of 450 clusters across different ages. Detailed charting efforts reveal extraordinary complexity where relationships among cell types reflect both spatial locations and developmental origins. Transcriptomically and developmentally related cell types can often be found in distant and diverse brain regions indicating that long-distance migration and dispersion is a common characteristic of nearly all classes of telencephalic GABAergic neurons. Additionally, we find various spatial dimensions of both discrete and continuous variations among related cell types that are correlated with gene expression gradients. Lastly, we find that cortical, striatal and some pallidal GABAergic neurons undergo extensive postnatal diversification, whereas septal and most pallidal GABAergic neuronal types emerge simultaneously during the embryonic stage with limited postnatal diversification. Overall, the telencephalic GABAergic cell type taxonomy can serve as a foundational reference for molecular, structural and functional studies of cell types and circuits by the entire community.

Membrane depolarization mediates both the inhibition of neural activity and cell-type-differences in response to high-frequency stimulation

Neuromodulation using high frequency (>1 kHz) electric stimulation (HFS) enables preferential activation or inhibition of individual neural types, offering the possibility of more effective treatments across a broad spectrum of neurological diseases. To improve effectiveness, it is important to better understand the mechanisms governing activation and inhibition with HFS so that selectivity can be optimized. In this study, we measure the membrane potential (Vm) and spiking responses of ON and OFF α-sustained retinal ganglion cells (RGCs) to a wide range of stimulus frequencies (100–2500 Hz) and amplitudes (10–100 µA). Our findings indicate that HFS induces shifts in Vm, with both the strength and polarity of the shifts dependent on the stimulus conditions. Spiking responses in each cell directly correlate with the shifts in Vm, where strong depolarization leads to spiking suppression. Comparisons between the two cell types reveal that ON cells are more depolarized by a given amplitude of HFS than OFF cells—this sensitivity difference enables the selective targeting. Computational modeling indicates that ion-channel dynamics largely account for the shifts in Vm, suggesting that a better understanding of the differences in ion-channel properties across cell types may improve the selectivity and ultimately, enhance HFS-based neurostimulation strategies.

PATH-02. THE IMPACT OF EVOLVING DIAGNOSTIC METHODOLOGY ON THE CLASSIFICATION AND OUTCOME OF CENTRAL NERVOUS SYSTEM TUMOURS IN CHILDHOOD: A RETROSPECTIVE COHORT STUDY

Abstract BACKGROUND Central nervous system tumours are the second most common childhood tumours and the largest cause of childhood cancer death in Australia. Tumours arise from many cell types within the developing and mature CNS, leading to a wide spectrum of clinical behaviour despite overlapping histological appearances. Emerging molecular testing methods allow more precise determination of cell lineage as well as genetic and epigenetic changes driving tumour development. Particularly since 2016, molecular findings have been incorporated into international classification schema, potentially impacting the frequency of individual diagnoses and their observed prognosis. METHODS We performed a retrospective cohort study including patients with newly diagnosed CNS tumours at Royal Children’s Hospital, Melbourne between 2011 and 2020 (total 473). The first and last 100 eligible patients in the data set were reviewed for diagnosis, survival and recorded use of molecular testing. Reviewed patients were assigned to one of two cohorts, Cohort 1 2011-2013 (pre-2016 WHO revision) or Cohort 2 2018-2020 (post-2016 WHO revision). RESULTS The frequency of diagnoses on biopsy rather than imaging alone was stable (81% Cohort 1 vs 83% Cohort 2). Molecular testing utilization increased markedly over time (10 molecular investigations in Cohort 1 vs 348 in Cohort 2). The incidence of astrocytic and oligodendroglial diagnoses was stable. The incidence of embryonal and neuronal/ mixed glial neuronal diagnoses increased in Cohort 2, and the incidence of ependymal diagnoses decreased (p=0.0122). There were no event free or overall survival differences between the whole of Cohorts 1 and 2, with numbers of individual diagnoses too small for meaningful comparisons over time. CONCLUSIONS Access to molecular testing has impacted on the pattern of reported diagnoses of central nervous system tumours in children. Although outcomes have not changed, it is hoped that over time, improved molecular understanding will lead to more effective therapy selection.

Poly(ferrocenylsilane)-Based Redox-Active Artificial Organelles for Biomimetic Cascade Reactions.

Artificial organelles serve as functional counterparts to natural organelles, which are primarily employed to artificially replicate, restore, or enhance cellular functions. While most artificial organelles exhibit basic functions, we diverge from this norm by utilizing poly(ferrocenylmethylethylthiocarboxypropylsilane) microcapsules (PFC MCs) to construct multifunctional artificial organelles through water/oil interfacial self-assembly. Within these PFC MCs, enzymatic cascades are induced through active molecular exchange across the membrane to mimic the functions of enzymes in mitochondria. We harness the inherent redox properties of the PFC polymer, which forms the membrane, to facilitate in-situ redox reactions similar to those supported by the inner membrane of natural mitochondria. Subsequent studies have demonstrated the interaction between PFC MCs and living cell including extended lifespans within various cell types. We anticipate that functional PFC MCs have the potential to serve as innovative platforms for organelle mimics capable of executing specific cellular functions.

MDB-16. CLINICAL AND MOLECULAR CHARACTERISTICS OF JAPANESE MEDULLOBLASTOMA, WNT-ACTIVATED

Abstract BACKGROUND Medulloblastomas are at least classified into four core subgroups, including WNT-activated, SHH-activated, group 3 and group 4. These molecular subgroups have been introduced in the latest WHO classification, which has become fundamental and indispensable. Medulloblastoma, WNT-activated (WNT-MB) accounts for about 10% of all medulloblastomas, and their prognosis is excellent, and it is desired to ensure that patients with WNT-MBs are diagnosed exactly. In this study, we have summarized results of molecular analysis of Japanese MBs collected by Japanese Pediatric Molecular Neuro-oncology Group (JPMNG) study, and analyzed clinical and molecular characteristics of WNT-MBs. METHODS We enrolled 267 cases of medulloblastoma from 44 centers participating in the JPMNG study since 2012.Central histopathological diagnosis was performed by three neuropathologists, and molecular genetic diagnosis was performed using molecular subgrouping, DNA sequencing (CTNNB1), and copy number analysis. RESULTS A total of 244 cases were finally analyzed, and the 4-subgroup classification was WNT: 39 (16%), SHH: 61 (25%), group3: 42 (17%), and group4: 102 (41.8%), respectively. The age at diagnosis was 3 years or older (6-40 years) in all cases, with no cases occurring in infants, the majority being children (6-18 years) (31/39: 79.5%), and 8 cases (8/39: 20.5%) in adults older than 18 years. Histological features were classic (CMB) in the majority of cases and large cell (LCA) type in only 2 cases. The positivity rate of nuclear β-catenin by Immunological staining was 9 out of 12 (75%) of the cases examined. While, CTNNB1mutation was positive in 39 of 39 cases (100%) and monosomy 6 was also positive in 35 of 35 cases (100%), respectively. The 5-year survival rate was 91.9%. CONCLUSIONS Clinical characteristics of Japanese WBT-MB were almost similar to those reported in other countries, which showed excellent prognosis, and their molecular genetic status were also identical with results from other ethnics.

MDB-70. SYSTEMATIC TRANSCRIPTOMIC ANALYSIS OF CHILDHOOD MEDULLOBLASTOMA IDENTIFIES M6A-LNCRNA SIGNATURES ASSOCIATED WITH PROGNOSIS, MOLECULAR SUBTYPE, AND IMMUNE CELL INFILTRATION

Abstract BACKGROUND Medulloblastoma (MB) is the predominant pediatric brain tumor, subdivided into four molecular subgroups, with group 3 and group 4 tumors posing significant clinical challenges due to poor prognosis and classification. This study investigates the roles of N6-methyladenosine (m6A) and long non-coding RNAs (lncRNAs) in MB, aiming to develop diagnostic and prognostic tools. METHODS We analyzed transcriptome from 1236 samples to identify m6A-associated lncRNA signature (M6LSig). A multivariate-cox analysis identified a gene signature significantly associated with overall survival (OS) of patients. Risk score was calculated based on the sum of weighted gene expression of these genes. Nomograms were calculated for predicting OS and PFS probability. We did feature selection with Boruta to identify minimum gene signature for subgroup classification. We performed correlation analysis of gene signature and risk score with immune-cell abundance. We depleted METTL3 and METTL14 in G3-MB cell-line (D425) to measure impact on cell proliferation and dysregulation of M6LSig genes as a result of loss of m6A. RESULTS Nomogram was constructed using risk score derived from 24 M6LSig significantly associated with OS and PFS of the patients. A 171 gene signature was identified that can accurately classify samples into one of four major subgroups of MB with more than 90% accuracy including between Grp3/Grp4 samples. M6LSig genes and risk score showed significant correlation with immune cell type abundance in MB tumors. Expression of CD155 and 4-1BB was significantly different between high and low risk samples. m6A writer genes knockdown in D425 cells resulted in decreased cell proliferation and upregulation of many M6LSig genes. CONCLUSION M6LSig based risk can accurately predict OS and PFS and 171 genes signature can classify patients into subgroups with more than 90% accuracy. Risk score can also be used for determining high risk versus low-risk patient and design tailor made immune-therapy for better survival.