Biology subjects

Ihrie, R. A.

Publications and source records attributed to Ihrie, R. A..

15 recordsLinked to original sources

bioRxiv2025

Spatial profiling of longitudinal glioblastoma reveals consistent changes in cellular architecture, post-treatment

Glioblastoma (GBM), the most aggressive adult brain cancer, comprises a complex tumour microenvironment (TME) with diverse cellular interactions driving progression and pathobiology. How these spatial patterns and interactions evolve with treatment remains unclear. Here, we apply imaging mass cytometry to analyse protein-level changes in paired pre- and post-treatment GBM samples from five patients. We find a significant post-treatment increase in normal brain cells alongside a reduction in vascular cells. Moreover, despite minimal overall change in cellular diversity, interactions among astrocytes, oligodendrocytes, and vascular cells increase post-treatment, suggesting reorganisation of the TME. The GBM TME cells form spatially organized layers driven by hypoxia pre-treatment, but this influence diminishes post-treatment, giving way to less organised layers with organisation driven by reactive astrocytes and lymphocytes. These findings provide insight into treatment-induced shifts in GBMs cellular landscape, highlighting aspects of the evolving TME that appear to facilitate recurrence and are, therefore, potential therapeutic targets. Key pointsO_LISpatial organisation in primary GBM consist of layers driven by the presence of hypoxia C_LIO_LIThe layers in recurrent GBM appear are driven more by the presence of reactive astrocytes C_LIO_LIIncreased cellular cross-talk in recurrent GBM presents novel therapeutic targets C_LI

cancer biology↗

bioRxiv2024

Temporally discordant chromatin accessibility and DNA demethylation define short and long-term enhancer regulation during cell fate specification

Epigenetic mechanisms govern the transcriptional activity of lineage-specifying enhancers; but recent work challenges the dogma that joint chromatin accessibility and DNA demethylation are prerequisites for transcription. To understand this paradox, we established a highly-resolved timeline of DNA demethylation, chromatin accessibility, and transcription factor occupancy during neural progenitor cell differentiation. We show thousands of enhancers undergo rapid, transient accessibility changes associated with distinct periods of transcription factor expression. However, most DNA methylation changes are unidirectional and delayed relative to chromatin dynamics, creating transiently discordant epigenetic states. Genome-wide detection of 5-hydroxymethylcytosine further revealed active demethylation begins ahead of chromatin and transcription factor activity, while enhancer hypomethylation persists long after these activities have dissipated. We demonstrate that these timepoint specific methylation states predict past, present and future chromatin accessibility using machine learning models. Thus, chromatin and DNA methylation collaborate on different timescales to mediate short and long-term enhancer regulation during cell fate specification.

genomics↗

bioRxiv2024

Human TSC2 Mutant Cells Exhibit Aberrations in Early Neurodevelopment Accompanied by Changes in the DNA Methylome

Tuberous Sclerosis Complex (TSC) is a debilitating developmental disorder characterized by a variety of clinical manifestations. While benign tumors in the heart, lungs, kidney, and brain are all hallmarks of the disease, the most severe symptoms of TSC are often neurological, including seizures, autism, psychiatric disorders, and intellectual disabilities. TSC is caused by loss of function mutations in the TSC1 or TSC2 genes and consequent dysregulation of signaling via mechanistic Target of Rapamycin Complex 1 (mTORC1). While TSC neurological phenotypes are well-documented, it is not yet known how early in neural development TSC1/2-mutant cells diverge from the typical developmental trajectory. Another outstanding question is the contribution of homozygous-mutant cells to disease phenotypes and whether such phenotypes are also seen in the heterozygous-mutant populations that comprise the vast majority of cells in patients. Using TSC patient-derived isogenic induced pluripotent stem cells (iPSCs) with defined genetic changes, we observed aberrant early neurodevelopment in vitro, including misexpression of key proteins associated with lineage commitment and premature electrical activity. These alterations in differentiation were coincident with hundreds of differentially methylated DNA regions, including loci associated with key genes in neurodevelopment. Collectively, these data suggest that mutation or loss of TSC2 affects gene regulation and expression at earlier timepoints than previously appreciated, with implications for whether and how prenatal treatment should be pursued.

developmental biology↗

bioRxiv2024

Velociraptor: Cross-Platform Quantitative Search Using Hallmark Cell Features

A key challenge for single cell discovery analysis is to identify new cell types, describe them quantitatively, and seek these novel cells in new studies often using a different platform. Over the last decade, tools were developed to address identification and quantitative description of cells in human tissues and tumors. However, automated validation of populations at the single cell level has struggled due to the cytometry fields reliance on hierarchical, ordered use of features and on platform-specific rules for data processing and analysis. Here we present Velociraptor, a workflow that implements Marker Enrichment Modeling in three cross-platform modules: 1) identification of cells specific to disease states, 2) description of hallmark features for each cell and population, and 3) searching for cells matching one or more hallmark feature sets in a new dataset. A key advance is that Velociraptor registers cells between datasets, including between flow cytometry and quantitative imaging using different, overlapping feature sets. Four datasets were used to challenge Velociraptor and reveal new biological insights. Working at the individual sample level, Velociraptor tracked the abundance of clinically significant glioblastoma brain tumor cell subsets and characterized the cells that predominate in recurrent tumors as a close match for rare, negative prognostic cells originally observed in matched pre-treatment tumors. In patients with inborn errors of immunity, Velociraptor identified genotype-specific cells associated with GATA2 haploinsufficiency. Finally, in cross-platform analysis of immune cells in multiplex imaging of breast cancer, Velociraptor sought and correctly identified memory T cell subsets in tumors. Different phenotypic descriptions generated by algorithms or humans were shown to be effective as search inputs, indicating that cell identity need not be described in terms of per-feature cutoffs or strict hierarchical analyses. Velociraptor thus identifies cells based on hallmark feature sets, such as protein expression signatures, and works effectively with data from multiple sources, including suspension flow cytometry, imaging, and search text based on known or theoretical cell features.

systems biology↗

bioRxiv2024

IL-8 Instructs Macrophage Identity in Lateral Ventricle Contacting Glioblastoma

Adult IDH-wildtype glioblastoma (GBM) is a highly aggressive brain tumor with no established immunotherapy or targeted therapy. Recently, CD32+ HLA-DRhi macrophages were shown to have displaced resident microglia in GBM tumors that contact the lateral ventricle stem cell niche. Since these lateral ventricle contacting GBM tumors have especially poor outcomes, identifying the origin and role of these CD32+ macrophages is likely critical to developing successful GBM immunotherapies. Here, we identify these CD32+ cells as M_IL-8 macrophages and establish that IL-8 is sufficient and necessary for tumor cells to instruct healthy macrophages into CD32+ M_IL-8 M2 macrophages. In ex vivo experiments with conditioned medium from primary human tumor cells, inhibitory antibodies to IL-8 blocked the generation of CD32+ M_IL-8 cells. Finally, using a set of 73 GBM tumors, IL-8 protein is shown to be present in GBM tumor cells in vivo and especially common in tumors contacting the lateral ventricle. These results provide a mechanistic origin for CD32+ macrophages that predominate in the microenvironment of the most aggressive GBM tumors. IL-8 and CD32+ macrophages should now be explored as targets in combination with GBM immunotherapies, especially for patients whose tumors present with radiographic contact with the ventricular-subventricular zone stem cell niche. - IL-8 is expressed by GBM cells and enriched in lateral ventricle-contacting tumors - M_IL-8 macrophages are CD32+ HLA-DR++ CD163+ CD206+ CD86- PD-L1- - M_IL-8 macrophages instructed with IL-8 or GBM conditioned medium match human glioblastoma associated macrophages - IL-8 is necessary for GBM tumor cells to generate M_IL-8 macrophages

cancer biology↗

bioRxiv2024

Multiparameter Quantitative Analyses of Diagnostic Cells in Brain Tissues from Tuberous Sclerosis Complex

The advent of high-dimensional imaging approaches offers innovative opportunities to molecularly characterize diagnostic cells in disorders that have previously relied on histopathological definitions. One example of such disorders is tuberous sclerosis complex (TSC), a developmental disorder characterized by systemic growth of benign tumors. Within resected brain tissues from patients with TSC, detection of abnormally enlarged balloon cells (BCs) is pathognomonic for this disorder. Though BCs can be identified by an expert neuropathologist, little is known about the specificity and broad applicability of protein markers for these cells, complicating classification of proposed BCs identified in experimental models of this disorder. Here, we report the development of a customized machine-learning workflow (Balloon Identifier; BAIDEN) that was trained to prospectively identify BCs in tissue sections using a histological stain compatible with high-dimensional cytometry. This approach was coupled to a custom antibody panel and 36-parameter imaging mass cytometry (IMC) to explore the expression of multiple previously proposed BC markers and develop a descriptor of BC features conserved across multiple tissue samples from patients with TSC. These findings comprise a toolbox and dataset for understanding the abundance, structure, and signaling activity of these histopathologically abnormal cells, and an example case of how such tools can be developed and applied within human tissues.

neuroscience↗

bioRxiv2023

Rescue of Impaired Blood-Brain Barrier in Tuberous Sclerosis Complex Patient Derived Neurovascular Unit

Tuberous sclerosis complex (TSC) is a multi-system genetic disease that causes benign tumors in the brain and other vital organs. The most debilitating symptoms result from involvement of the central nervous system and lead to a multitude of severe symptoms including seizures, intellectual disability, autism, and behavioral problems. TSC is caused by heterozygous mutations of either the TSC1 or TSC2 gene. Dysregulation of mTOR kinase with its multifaceted downstream signaling alterations is central to disease pathogenesis. Although the neurological sequelae of the disease are well established, little is known about how these mutations might affect cellular components and the function of the blood-brain barrier (BBB). We generated disease-specific cell models of the BBB by leveraging human induced pluripotent stem cell and microfluidic cell culture technologies. Using these microphysiological systems, we demonstrate that the BBB generated from TSC2 heterozygous mutant cells shows increased permeability which can be rescued by wild type astrocytes and with treatment with rapamycin, an mTOR kinase inhibitor. Our results further demonstrate the utility of microphysiological systems to study human neurological disorders and advance our knowledge of the cell lineages contributing to TSC pathogenesis.

neuroscience↗

bioRxiv2023

Jedi-1/MEGF12-mediated phagocytosis controls the pro-neurogenic properties of microglia in the ventricular-subventricular zone

Microglia are the primary phagocytes in the central nervous system and are responsible for clearing dead cells generated during development or disease. The phagocytic process shapes the phenotype of the microglia, which affects the local environment. A unique population of microglia reside in the ventricular-subventricular zone (V-SVZ) of neonatal mice, but how they influence this neurogenic niche is not well-understood. Here, we demonstrate that phagocytosis creates a pro-neurogenic microglial phenotype in the V-SVZ and that these microglia phagocytose apoptotic cells via the engulfment receptor Jedi-1. Deletion of Jedi-1 decreases apoptotic cell clearance, triggering the development of a neuroinflammatory phenotype, reminiscent of neurodegenerative and-age-associated microglia, that reduces neural precursor proliferation via elevated interleukin (IL)-1{beta} signaling; inhibition of IL-1 receptor rescues precursor proliferation in vivo. Together, these results reveal a critical role for Jedi-1 in connecting microglial phagocytic activity to a phenotype that promotes neurogenesis in the developing V-SVZ. Graphical Abstract. Jedi-1-dependent phagocytosis supports neurogenesis via suppression of microglial inflammatory pathway activationTop: Wild-type Proliferative-zone-Associated Microglia (PAMs) (cyan) use the engulfment receptor Jedi-1 ( Jedi) to engulf apoptotic cells (yellow) in the neurogenic ventricular-subventricular zone (V-SVZ) of the early postnatal brain. Jedi activation supports neural precursor cell (NPC) proliferation and the generation of new neurons. Bottom: Deletion of Jedi reduces microglial phagocytosis and transforms PAMs into Disease-associated Inflammatory Microglia (DIMs) characterized by the upregulation of canonical inflammatory genes and core DIM markers iden ified in the aging and neurodegenerative brain (Nlrp3, NLR family pyrin domain-containing 3; Tnf, tumor necrosis factor; Ccl4, C-C chemokine ligand 4 (also called macrophage inflammatory protein 1{beta}); Ccr5, C-C chemokine receptor type 5). Increased interleukin-1{beta} (IL-1{beta}) synthesis, release, and signaling in the Jedi-null V-SVZ reduces NPC proliferation and newborn neuron number. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=119 SRC="FIGDIR/small/531012v1_ufig1.gif" ALT="Figure 1"> View larger version (34K): org.highwire.dtl.DTLVardef@16b9785org.highwire.dtl.DTLVardef@a8be50org.highwire.dtl.DTLVardef@66b726org.highwire.dtl.DTLVardef@1825d8b_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIThe engulfment receptor Jedi-1 is expressed by microglia in the neonatal ventricular-subventricular zone (V-SVZ) neurogenic niche. C_LIO_LIJedi-1 knockout microglia have decreased engulfment ability, resulting in accumulation of dead cells in the V-SVZ. C_LIO_LILoss of Jedi-1 leads to a neuroinflammatory phenotype in microglia that is characteristic of neurodegenerative and age-associated microglia. C_LIO_LIMicroglial-specific loss of Jedi-1 reduces neurogenesis, which is rescued in vivo by inhibition of interleukin-1{beta} signaling. C_LI

neuroscience↗

bioRxiv2023

Dephosphorylation of 4EBP1/2 Induces Prenatal Neural Stem Cell Quiescence

A limiting factor in the regenerative capacity of the adult brain is the abundance and proliferative ability of neural stem cells (NSCs). Adult NSCs are derived from a subpopulation of embryonic NSCs that temporarily enter quiescence during mid-gestation and remain quiescent until postnatal reactivation. Here we present evidence that the mechanistic/mammalian target of rapamycin (mTOR) pathway regulates quiescence entry in embryonic NSCs of the developing forebrain. Throughout embryogenesis, two downstream effectors of mTOR, p-4EBP1/2 T37/46 and p-S6 S240/244, were mutually exclusive in NSCs, rarely occurring in the same cell. While 4EBP1/2 was phosphorylated in stem cells undergoing mitosis at the ventricular surface, S6 was phosphorylated in more differentiated cells migrating away from the ventricle. Phosphorylation of 4EBP1/2, but not S6, was responsive to quiescence induction in cultured embryonic NSCs. Further, inhibition of p-4EBP1/2, but not p-S6, was sufficient to induce quiescence. Collectively, this work offers new insight into the regulation of quiescence entry in embryonic NSCs and, thereby, correct patterning of the adult brain. These data suggest unique biological functions of specific posttranslational modifications and indicate that the preferential inhibition of such modifications may be a useful therapeutic approach in neurodevelopmental diseases where NSC numbers, proliferation, and differentiation are altered.

developmental biology↗

bioRxiv2022

Non-Canonical Functions of a Mutant TSC2 Protein in Mitotic Division

Tuberous Sclerosis Complex (TSC) is a debilitating neurodevelopmental disorder characterized by a variety of clinical manifestations including epilepsy, autism, and intellectual disability. TSC is caused by mutations in the TSC1 or TSC2 genes, which encode the hamartin/tuberin proteins respectively. These proteins function as a heterodimer that negatively regulates mechanistic Target of Rapamycin Complex 1 (mTORC1). TSC research has focused on the effects of mTORC1, a critical signaling hub, on regulation of diverse cell processes including metabolism, cell growth, translation, and neurogenesis. However, non-canonical functions of TSC2 are not well studied, and the potential disease-relevant biological mechanisms are not well understood. We observed aberrant multipolar mitotic division, a novel phenotype, in TSC2 mutant iPSCs. The multipolar phenotype is not meaningfully affected by treatment with mTORC1 inhibition, suggesting that multipolar division is an mTORC1-independent phenotype. We further observed dominant negative activity of the mutant form of TSC2 in producing the multipolar division phenotype. These data expand the knowledge of TSC2 function and pathophysiology which will be highly relevant to future treatments for patients with TSC. HighlightsO_LINovel multipolar division in patient-derived iPSCs with mutant form of tuberin, TSC2 encoded protein C_LIO_LIMutant tuberin may act in a dominant negative, mTORC1-independent manner C_LI eTOCTuberous sclerosis complex (TSC) is a disorder caused by mutations in TSC1 or TSC2 genes leading to mTORC1 hyperactivity. Chalkley and colleagues found that a mutant microdeletion allele of TSC2 causes multipolar division in human induced pluripotent stem cells. Chalkley and colleagues also found that the multipolar division from mutant TSC2 may have a dominant negative mechanism and be mTOR1-independent.

cell biology↗

bioRxiv2022

GBMdeconvoluteR accurately infers proportions of neoplastic and immune cell populations from bulk glioblastoma transcriptomics data

The biological and clinical impact of neoplastic and immune cell type ratios in the glioblastoma (GBM) tumour microenvironment is being realised. Characterising and quantifying cell types within GBMs at scale will facilitate a better understanding of the association between the cellular landscape and tumour phenotypes or clinical correlates. This study aimed to develop a tool that can deconvolute immune and neoplastic cells within the GBM tumour microenvironment from bulk RNA sequencing data. We developed an IDH wild-type (IDHwt) GBM specific single immune cell reference dataset, from four independent studies, consisting of B cells, T cells, NK cells, microglia, tumour associated macrophages, monocytes, mast and DC cells. We used this alongside an existing neoplastic single cell-type dataset consisting of astrocyte-like, oligodendrocyte- and neuronal-progenitor like and mesenchymal GBM cancer cells to create both marker and gene signature matrix-based deconvolution tools. We then applied single-cell resolution imaging mass cytometry (IMC) to ten IDHwt GBM samples, five paired primary and recurrent tumours, in parallel with these tools to determine which performed best. Marker based gene expression deconvolution using GBM tissue specific markers, which we have packaged as GBMdeconvoluteR, gave the most accurate results. The correlation between immune cell quantification by IMC and by GBMdeconvoluteR for primary IDHwt GBM samples was 0.52 (Pearsons P=7.8x10-3) and between neoplastic cell quantification by IMC and by GBMdeconvoluteR was 0.75 (Pearsons P=1.2x10-3). We applied GBMdeconvoluteR to bulk GBM RNAseq data from The Cancer Genome Atlas (TCGA) and were able to recapitulate recent findings from multi-omics single cell studies with regards associations between mesenchymal GBM cancer cells and both lymphoid and myeloid cells. Furthermore, we were able to expand upon this to show that these associations are stronger in patients with worse prognosis. GBMdeconvoluteR is accessible online at https://gbmdeconvoluter.leeds.ac.uk. Key pointsGBMdeconvoluteR is a glioblastoma-specific cellular deconvolution tool. When applied to bulk GBM RNAseq data, it accurately quantifies the neoplastic and immune cells in that tumour. It is available online at https://gbmdeconvoluter.leeds.ac.uk

cancer biology↗

bioRxiv2022

Alignment, Segmentation and Neighborhood Analysis in Cyclic Immunohistochemistry Data Using CASSATT

Cyclic immunohistochemistry (cycIHC) uses sequential rounds of colorimetric immunostaining and imaging for quantitative mapping of location and number of cells of interest. In addition, cycIHC benefits from the speed and simplicity of brightfield microscopy for data collection, making the collection of entire tissue sections and slides possible at a trivial cost compared to other high dimensional imaging modalities. However, large cycIHC datasets (greater than 50 GB) currently require an expert data scientist to concatenate separate open-source tools for each step of image pre-processing, registration, and segmentation, or the use of proprietary software. Here, we present a unified and user-friendly pipeline for processing, aligning, and analyzing cycIHC data - Cyclic Analysis of Single-Cell Subsets and Tissue Territories (CASSATT). CASSATT registers scanned slide images across all rounds of staining, segments individual nuclei, and measures marker expression on each detected cell. Beyond straightforward single cell data analysis outputs, CASSATT explores the spatial relationships between cell populations. By calculating the logodds of interaction frequencies between cell populations within tissues and tissue regions, this pipeline helps users identify populations of cells that interact - or do not interact - at frequencies that are greater than those occurring by chance. It also identifies specific neighborhoods of cells based on the assortment of neighboring cell types that surround each cell in the sample. The presence and location of these neighborhoods can be compared across slides or within distinct regions within a tissue. CASSATT was first tested using a newly generated cycIHC dataset consisting of six GBM tissue sections processed through eight cycles of AEC based IHC staining. Further validation was completed on a previously published lung cancer tissue microarray dataset consisting of 107 cores processed through eighteen cycles of staining and imaging. CASSATT is a fully open-source workflow tool developed to process cycIHC data and will allow greater utilization of this powerful staining technique.

bioinformatics↗

bioRxiv2022

Immune cell regulation in stem cell niche contacting glioblastomas.

Glioblastomas (GBM) are tumors for which immune-targeted therapies have failed to show clinical benefit and for which few biomarkers provide context for meaningful therapeutic stratification. Radiographic contact of GBM tumors with the lateral ventricle stem cell niche correlates with worse patient prognosis; however, the extent to which proximity to the ventricle impacts antitumor immunity remains unknown. We demonstrate that T cell checkpoint receptor expression is elevated in ventricle-contacting GBM as is the abundance of a specific, suppressive CD32+CD44+HLADhigh myeloid population suggesting a distinct immunoregulatory influence on antitumor immunity in proximity to the lateral ventricle. Phospho-specific mass cytometric profiling revealed extensively impaired immune signaling in ventricle-contacting GBM in response to inflammatory cytokine stimulation, further supporting a suppressive milieu influencing immunity at the lateral ventricle. Collectively, we identify a regulatory impact of ventricle contact on antitumor immunity in the brain, and reveal novel clinically targetable mechanisms of immunomodulation in patients with glioblastoma. Significance StatementWe demonstrate that the immune microenvironment of glioblastoma tumors contacting the lateral ventricle differs from non-contacting tumors. This work connects immune-biology to a radiographically detectable feature, the lateral ventricle, and highlights non-invasive imaging as a means to identify targetable immune features in glioblastoma tumors.

immunology↗

bioRxiv2020

Creation and validation of 3D-printed head molds for stereotaxic injections of neonatal mouse brains

BackgroundAn increasing number of rodent model systems use injection of DNA or viral constructs in the neonatal brain. However, approaches for reliable positioning and stereotaxic injection at this developmental stage are limited, typically relying on handheld positioning or molds that must be re-aligned for use in a given laboratory. New methodA complete protocol and open source software pipeline for generating 3D-printed head molds derived from a CT scan of a neonatal mouse head cast, together with a universal adapter that can be placed on a standard stereotaxic stage. ResultsA series of test injections with adenovirus encoding red fluorescent protein were conducted using original clay molds and newly generated 3D printed molds. Several metrics were used to compare spread and localization of targeted injections. Comparison with existing methodsThe new method of head mold generation gave comparable results to the field standard, but also allowed the rapid generation of additional copies of each head mold with standardized positioning of the head each time. ConclusionsThis 3D printing pipeline can be used to rapidly develop a series of head molds with standardized injection coordinates across multiple laboratories. More broadly, this pipeline can easily be adapted to other perinatal ages or species.

neuroscience↗

bioRxiv2019

High risk glioblastoma cells revealed by machine learning and single cell signaling profiles

Recent developments in machine learning implemented dimensionality reduction and clustering tools to classify the cellular composition of patient-derived tissue in multi-dimensional, single cell studies. Current approaches, however, require prior knowledge of either categorical clinical outcomes or cell type identities. These algorithms are not well suited for application in tumor biology, where clinical outcomes can be continuous and censored and cell identities may be novel and plastic. Risk Assessment Population IDentification (RAPID) is an unsupervised, machine learning algorithm that identifies single cell phenotypes and assesses clinical risk stratification as a continuous variable. Single cell mass cytometry evaluated 34 different phospho-proteins, transcription factors, and cell identity proteins in tumor tissue resected from patients bearing IDH wild-type glioblastomas. RAPID identified and characterized multiple biologically distinct tumor cell subsets that independently and continuously stratified patient outcome. RAPID is broadly applicable for single cell studies where atypical cancer and immune cells may drive disease biology and treatment responses.

cancer biology↗