Biology subjects

Canoll, P.

Publications and source records attributed to Canoll, P..

At least 19 recordsLinked to original sources

bioRxiv2026

Expanding the therapeutic window of bacteria for cancer therapy

Engineered bacteria enable self-amplifying therapeutic delivery and immune activation, yet these same features drive systemic toxicity and off-target growth. Here, we create a "tumorobe" that overcomes these limitations by integrating reduced immunogenicity, tumour-restricted growth, and enhanced payload release. Specifically, we identify lipopolysaccharide modifications that reduce immunogenicity while preserving fitness, restrict growth to tumours using a hypoxia-lactate "AND" gate, and enhance extracellular payload release via lipoprotein deletion. This strain expands the maximum tolerated dose in a route-dependent manner by up to 1,000-fold. Across multiple murine models, including colorectal cancer and glioblastoma, the strain drives robust antitumour efficacy as both a standalone therapeutic and an immunotherapeutic delivery platform, achieving durable complete responses in up to 75% of colorectal tumours when armed with a cytokine payload. Together, these results establish a broadly applicable platform for safe and effective bacterial cancer therapy.

synthetic biology↗

bioRxiv2026

Microtubule Acetylation Regulates the Malignant Phenotype of Glioblastoma and is a Promising Therapeutic Target

Glioblastoma (GBM) is universally lethal despite decades of research to find effective treatments. This highlights the need to identify druggable targets essential for sustaining the malignant phenotype but dispensable for normal tissue. We propose that the enzyme -tubulin acetyl transferase (ATAT1) meets these criteria. ATAT1 acetylates -tubulin at lysine 40, which increases microtubule stability and promotes microtubule-based transport. While ATAT1 knockout mice have only a very mild phenotype, ATAT1 suppression in GBM has multiple therapeutic effects by reducing tumor invasion, proliferation, and therapeutic resistance. These translate not only into improved survival with ATAT1 targeting by itself, but also into synergy when ATAT1 deletion is combined with FDA approved therapies. This study strongly supports our conclusion that ATAT1 is a promising therapeutic target in GBM.

Cancer Biology↗

bioRxiv2026

Paired Tumor Biopsies Reveal Spatiotemporal Myeloid Remodeling After Local Chemotherapy in Glioblastoma

BackgroundStandard-of-care chemotherapy for glioblastoma induces inflammation that may contribute to disease recurrence. Although recurrent tumors are enriched with myeloid cells, the early cellular response to chemotherapy and the mechanisms that initiate this inflammatory remodeling remain poorly understood. In particular, it is unknown how neoplastic and tumor-associated myeloid populations respond during the immediate post-treatment period, and whether tumor-associated myeloid cells directly experience chemotherapy-induced genotoxic stress that contributes to inflammatory state transitions. MethodsWe performed sequencing-based analysis of neoplastic and immune populations following topotecan exposure using multiple complementary model systems and time points. We first analyzed MRI-localized, paired pre- and post-treatment biopsies from a first-in-human trial of 28-day convection enhanced delivery (CED) of topotecan (n=5) using cell-type-deconvolved bulk-RNA-sequencing and immunofluorescence. We then treated syngeneic murine gliomas using an in vivo model of CED-topotecan and measured acute 3-day and 7-day treatment responses by single cell RNA-sequencing. We additionally conducted sequencing analysis of patient-derived slice cultures and in vitro human microglial and glioma cell lines following 24-hour topotecan treatment. ResultsIn paired human biopsies, CED-topotecan induced spatially restricted transcriptional remodeling within the infusion zone, characterized by suppression of proliferative tumor programs and enrichment of inflammatory, interferon, hypoxia, and mesenchymal signatures. Cell-type deconvolution and immunofluorescence linked this response to myeloid remodeling, including enrichment of monocyte-derived tumor-associated macrophage states, increased MARCO-positive myeloid populations, and pH2AX-positive genotoxic stress within Iba1-positive myeloid cells. In the murine CED model, topotecan prolonged survival and reduced tumor cellularity, while also inducing inflammatory and DNA-damage programs in tumor-associated macrophages that evolved by 7-days toward hypoxia, angiogenesis, TGF-{beta} signaling, and mesenchymal/tissue-remodeling programs. Human slice culture and in vitro microglial systems confirmed stress-coupled inflammatory and DNA-damage responses in human myeloid cells. ConclusionsChemotherapy exposure induces a spatially structured inflammatory myeloid response characterized by early genotoxic stress and inflammatory activation, with later emergence of mesenchymal and tissue-remodeling macrophage programs. Across model systems, our analysis supports a model in which chemotherapy-associated damage in both tumor and myeloid cells contributes to an evolving inflammatory microenvironment after treatment.

neuroscience↗

bioRxiv2026

Electrophysiologically Targeted Biopsies Reveal the Transcriptional Landscape of Focal Epilepsy

Up to 30% of patients with epilepsy have intractable seizures, yet the mechanisms of focal ictogenesis remain unclear. Tissue involvement in ictal regions is heterogeneous, with different regions playing distinct roles in ictogenesis, seizure propagation, and resistance to spread. These roles are reflected in electrophysiologic differences between the seizure focus and the ictal penumbra, where evidence of synaptic spread is present but excitatory firing is constrained by largely intact inhibition. Investigation of the disruption of the normal interplay between excitatory and inhibitory activity, thought to underlie ictogenesis across a range of epilepsy etiologies, is limited by network complexity and cellular heterogeneity in human tissue samples. In this study, we relate cellular and molecular alterations to excitatory-inhibitory disruption in network dynamics defined by electrophysiologic features. This work may aid in the identification of clinically relevant tissue biomarkers and support novel preclinical therapeutic approaches for treatment-resistant focal epilepsy disorders. We developed a novel intracranial EEG guided, MRI-localized approach to sample paired biopsies from 11 patients with drug-resistant focal epilepsy with diverse etiologies, which were then studied using single-nucleus RNA sequencing (snRNAseq) and immunohistochemistry (IHC). EEG recorded from stereotactically implanted depth arrays (sEEG) was used to identify regions of epileptic involvement, based on findings from prior simultaneous clinical and microelectrode recordings. This approach addresses the intrinsic heterogeneity due to etiology and cortical architecture through paired, within-patient comparisons. We identified distinct cell-type specific transcriptional signatures that differentiate cellular populations in the seizure focus and ictal penumbra in intractable focal epilepsies. Our findings provide a link between tissue composition and gene expression that correlate with electrographic features in a heterogeneous seizure landscape. Our findings support common pathways of seizure generation and spread that are conserved across disease etiologies. Relative depletion of interneuron populations in the seizure focus supports the hypothesis of disrupted inhibition as a driver of epileptiform activity in the seizure focus. The enrichment of plasticity-associated gene signatures in the penumbra suggests a complex interaction of these regions with the seizure focus, as well as the role of the penumbra in enabling or limiting seizure expansion. This study provides a novel methodology for tissue sampling in epilepsy and uncovers biologically relevant tissue signatures that provide grounds for future work in targeting cellular and molecular alterations present in focal epilepsies.

neuroscience↗

bioRxiv2026

IDH-mutant inhibitors enhance the sensitivity of IDH1-mutant gliomas to cysteine-methionine deprivation and ferroptosis

Cysteine is essential for synthesizing glutathione, the brains main antioxidant, and cysteine deprivation can trigger ferroptosis. Here, using a new mouse model of IDH1-mutant glioma that recapitulates the characteristics of human IDH1-mutant low-grade gliomas, we demonstrate that IDH1-mutant glioma cells are significantly more vulnerable to cysteine deprivation alone or in combination with the ferroptosis inducer RSL3, compared to IDH1-wildtype glioma cells. In addition, treatments with the IDH-mutant inhibitors vorasidenib and ivosidenib further sensitize the cells to ferroptosis. Metabolomics analysis reveals that IDH1-mutant cells have altered cysteine and methionine metabolism with deficiency in transsulfuration, which is further exacerbated by cysteine-methionine deprivation and IDH-mutant inhibitors. Furthermore, dietary cysteine-methionine deprivation alone or in combination with convection-enhanced delivery of RSL3 or ivosidenib in vivo significantly prolongs survival of IDH1-mutant tumor-bearing mice. Our findings suggest that targeting cysteine and methionine metabolism in combination with IDH-mutant inhibition provides promising therapeutic strategies for IDH1-mutant gliomas.

cancer biology↗

bioRxiv2025

Convection-enhanced delivery of dexamethasone in glioma suppresses myeloid inflammation while avoiding systemic toxicities

Dexamethasone is widely used to control cerebral edema and inflammation in glioblastoma, but its benefits are limited by systemic toxicities and adverse prognostic associations. We evaluated local administration of dexamethasone via convection-enhanced delivery (CED) to maximize intratumoral anti-inflammatory effects by increasing local corticosteroid exposure while minimizing systemic exposure. In two glioma mouse models, continuous intraparenchymal infusion of dexamethasone was well tolerated with no adverse effects. Pharmacokinetic analyses supported preferential intratumoral distribution and reduced systemic exposure with CED compared with systemic dosing. Single-nucleus RNA sequencing (snRNA-seq) and immunohistochemistry showed attenuation of glioma-associated inflammation with downregulation of reactive microglial/macrophage programs and reduced tumor-infiltrating myeloid cells with a morphology consistent with a less activated state. Experiments in human induced pluripotent stem cell (iPSC)-derived microglia confirmed that dexamethasone directly suppresses inflammatory gene expression, indicating a conserved mechanism across species. This inflammatory suppression was recapitulated in both immortalized microglial (HMC3) and macrophage (THP1) cell lines. These findings suggest that localized dexamethasone delivered by CED reprograms the glioma immune microenvironment and achieves control of inflammation without the systemic adverse effects associated with standard systemic dexamethasone therapy. This clinically translatable strategy may improve symptom management and provide a platform for integrating local immunomodulation with future glioblastoma therapies.

cancer biology↗

bioRxiv2025

A proteogenomic tool uncovers protein markers for human microglial states

Human microglial heterogeneity has been largely described using transcriptomic data. Here, we introduce a microglial proteomic data resource and a Cellular Indexing of Transcriptomes and Epitopes by Sequencing panel enhanced with antibodies targeting 17 microglial cell surface proteins (mCITE-Seq). We evaluated mCITE-Seq on HMC3 microglia-like cells, induced-pluripotent stem cell-derived microglia (iMG), and freshly isolated primary human microglia. We identified novel protein microglial markers such as CD51 and relate expression of 101 cell surface proteins to transcriptional programs. This results in the identification and validation of three protein marker combinations with which to purify microglia enriched with each of 23 transcriptional programs; for example, CD49D, HLA-DR and CD32 enrich for GPNMBhigh ("disease associated") microglia. Further, we identify and validate proteins - SIRPA, PDPN and CD162 - that differentiate microglia from infiltrating macrophages. The mCITE-Seq panel enables the transition from RNA-based classification and facilitates the functional characterization and harmonization of model systems.

neuroscience↗

bioRxiv2025

A therapeutic role for a regulatory glucose transporter1 (Glut1)-associated natural antisense transcript

The mammalian brain relies primarily on glucose for its energy needs. Delivery of this nutrient to the brain is mediated by the glucose transporter-1 (Glut1) protein. Low Glut1 thwarts glucose entry into the brain, causing an energy crisis and, triggering, in one instance, the debilitating neurodevelopmental condition - Glut1 deficiency syndrome (Glut1DS). Current treatments for Glut1DS are sub-optimal, as none address the root cause - low Glut1 - of the condition. Levels of this transporter must respond rapidly to the brains changing energy requirements. This necessitates fine-tuning its expression. Here we describe a long-noncoding RNA (lncRNA) antisense to Glut1 and show that it is involved in such regulation. Raising levels of the lncRNA had a concordant effect on Glut1 in cultured human cells and transgenic mice; reducing levels elicited the opposite effect. Delivering the lncRNA to Glut1DS model mice via viral vectors induced Glut1 expression, enhancing brain glucose levels to mitigate disease. Direct delivery of such a lncRNA to combat disease has not been reported previously and constitutes a unique therapeutic paradigm. Moreover, considering the importance of maintaining homeostatic Glut1 levels, calibrating transporter expression via the lncRNA could become broadly relevant to the myriad conditions, including Alzheimers disease, wherein Glut1 concentrations are perturbed.

neuroscience↗

bioRxiv2025

A factor-based analysis of individual human microglia uncovers regulators of an Alzheimer-related transcriptional signature

Human microglial heterogeneity is only beginning to be appreciated at the molecular level. Here, we present a large, single-cell atlas of expression signatures from 441,088 live microglia broadly sampled across a diverse set of brain regions and neurodegenerative and neuroinflammatory diseases obtained from 161 donors sampled at autopsy or during a neurosurgical procedure. Using single-cell hierarchical Poisson factorization (scHPF), we derived a 23-factor model for continuous gene expression signatures across microglia which capture specific biological processes (e.g., metabolism, phagocytosis, antigen presentation, inflammatory signaling, disease-associated states). Using external datasets, we evaluated the aspects of microglial phenotypes that are encapsulated in various in vitro and in vivo microglia models and identified and replicated the role of two factors in human postmortem tissue of Alzheimers disease (AD). Further, we derived a complex network of transcriptional regulators for all factors, including regulators of an AD-related factor enriched for the mouse disease-associated microglia 2 (DAM2) signature: ARID5B, CEBPA, MITF, and PPARG. We replicated the role of these four regulators in the AD-related factor and then designed a multiplexed MERFISH panel to assess our microglial factors using spatial transcriptomics. We find that, unlike cells with high expression of the interferon-response factor, cells with high expression of the AD DAM2-like factor are widely distributed in neocortical tissue. We thus propose a novel analytic framework that provides a taxonomic approach for microglia that is more biologically interpretable and use it to uncover new therapeutic targets for AD.

neuroscience↗

bioRxiv2025

High throughput identification of genetic regulators of microglial inflammatory processes in Alzheimer's disease

Genome-wide association studies (GWAS) have identified over a hundred genetic risk factors for Alzheimers disease (AD), many of which are predominantly expressed in microglia. However, the pathogenic role for most of them remains unclear. To systematically investigate how AD GWAS variants influence human microglial inflammatory responses, we conducted CRISPR inhibition (CRISPRi) screens targeting 119 AD GWAS hits in hiPSC-derived microglia (iMGLs) and used the production of reactive oxygen species (ROS) in response to the viral mimic poly(I:C) as a functional readout. Top hits whose knockdown either increased or decreased ROS levels in response to poly(I:C) were further analyzed using CROP-seq to integrate CRISPRi with single-cell RNA sequencing (scRNA-seq). This analysis identified 9 unique microglial clusters, including a poly(I:C)-driven inflammatory cluster 2. Emerging evidence supports a pathogenic role of viral infections in AD and cross comparison of our scRNA-seq data with iMGLs xenotransplanted into an AD mouse model shows significant overlap between our clusters and AD-relevant microglial clusters. Knockdown of MS4A6A and EED, which resulted in elevated ROS production in the presence of poly(I:C), increased the proportion of cluster 2 cells and induced functionally related changes in gene expression. In addition, KD of MS4A6 led to a reduction in the proportion of iMGLs in the DAM (disease associated microglia) cluster under all conditions, suggesting that this gene may modulate the DAM response. In contrast, KD of INPP5D or RAPEP1 which lead to low levels of ROS in the presence of poly(I:C), did not significantly affect the proportion of cells in cluster 2 but rather shaped the inflammatory response. This included the upregulation of an HLA-associated inflammatory cluster (cluster 6) by INPP5D knockdown under all conditions, independent of poly(I:C) stimulation. Importantly, KD of INPP5D or RAPEP1 had many shared differentially expressed genes (DEGs) under both vehicle and poly(I:C) treated conditions. Overall, our findings demonstrate that despite the diverse biological functions of AD GWAS variants, they converge functionally to regulate human microglial states and shape inflammatory responses relevant to AD pathology.

neuroscience↗

bioRxiv2024

Splicing dysregulation in glioblastoma alters the function of cell migration-related genes

Glioblastoma (GBM) has a poor prognosis with a high recurrence and low survival rate. Previous RNA-seq analyses have revealed that alternative splicing (AS) plays a role in GBM progression. Here, we present a novel AS analysis method (Semi-Q) and describe its use to identify GBM-specific AS events. We analyzed RNA-seq data from normal brain (NB), normal human astrocytes (NHAs) and GBM samples, and found that comparison between NHA and GBM was especially informative. Importantly, this analysis revealed that genes encoding cell migration-related proteins, including filamins (FLNs) and actinins (ACTNs), were among those most affected by differential AS. Functional assays revealed that dysregulated AS of FLNA, B and C transcripts produced protein isoforms that not only altered transcription of cell proliferation-related genes but also led to enhanced cell migration, resistance to cell death and/or mitochondrial respiratory function, while a dysregulated AS isoform of ACTN4 enhanced cell migration. Together, our results indicate that cell migration and actin cytoskeleton-related genes are differentially regulated by AS in GBM, supporting a role for AS in facilitating tumor growth and invasiveness.

molecular biology↗

bioRxiv2024

MAPK/ERK signaling in gliomas modulates interferon responses, T cell recruitment, enhances tumor-microglia crosstalk, and drives immune checkpoint blockade efficacy.

BackgroundGlioblastoma (GB) remains a formidable challenge in neuro-oncology, with immune checkpoint blockade (ICB) showing limited efficacy in unselected patients. We previously recently established that MAPK/ERK signaling is associated with overall survival following anti-PD-1 and anti-CTLA-4 treatment in recurrent GB. However, the causal relationship between MAPK/ERK signaling and susceptibility to ICB, as well as the mechanisms underlying this association, remain poorly understood. MethodWe conducted in vivo kinome-wide CRISPR/Cas9 screenings in murine gliomas to identify key regulators of susceptibility to anti-PD-1 and CD8+ T cell responses and performed survival studies to validate the most relevant genes. Additionally, paired single cell RNA- sequencing (scRNA-seq) with p-ERK staining, spatial transcriptomics on GB samples, and ex-vivo slice culture of a BRAFV600E mutant GB tumor treated with BRAFi/MEKi were used to determine the causal relationship between MAPK signaling, tumor cell immunogenicity, and modulation of microglia phenotype. ResultsCRISPR/Cas9 screens identified the MAPK pathway, particularly the RAF-MEK-ERK pathway, as the most critical modulator of glioma susceptibility to CD8+ T cells, and anti-PD-1 across all kinases. Experimentally-induced ERK phosphorylation in gliomas enhanced survival with ICB treatment, led to durable anti-tumoral immunity upon re-challenge and memory T cell infiltration in long-term survivors. Elevated p-ERK in glioma cells correlated with increased interferon responses, antigen presentation and T cell infiltration in GB. Moreover, spatial transcriptomics and scRNA-seq analysis revealed the modulation of interferon responses by the MAPK/ERK pathway in BRAFV600E human GB cells with ERK1/2 knockout and in slice cultures of human BRAFV600E GB tissue. Notably, BRAFi/MEKi treatment disrupted the interaction between tumor cells and tumor-associated macrophages/microglia in slice cultures from BRAFV600E mutant GB. ConclusionOur data indicate that the MAPK/ERK pathway is a critical regulator of GB cell susceptibility to anti-tumoral immunity, modulating interferon responses, and antigen-presentation in glioma cells, as well as tumor cell interaction with microglia. These findings not only elucidate the mechanistic underpinnings of immunotherapy resistance in GB but also highlight the MAPK/ERK pathway as a promising target for enhancing immunotherapeutic efficacy in this challenging malignancy.

cancer biology↗

bioRxiv2024

Resistance to Spindle Inhibitors in Glioblastoma Depends on STAT3 and Therapy Induced Senescence

While mitotic spindle inhibitors specifically kill proliferating tumor cells without the toxicities of microtubule poisons, resistance has limited their clinical utility. Treating glioblastomas with the spindle inhibitors ispinesib, alisertib, or volasertib creates a subpopulation of therapy induced senescent cells that resist these drugs by relying upon the anti-apoptotic and metabolic effects of activated STAT3. Furthermore, these senescent cells expand the repertoire of cells resistant to these drugs by secreting an array of factors, including TGF{beta}, which induce proliferating cells to exit mitosis and become quiescent--a state that also resists spindle inhibitors. Targeting STAT3 restores sensitivity to each of these drugs by depleting the senescent subpopulation and inducing quiescent cells to enter the mitotic cycle. These results support a therapeutic strategy of targeting STAT3-dependent therapy-induced senescence to enhance the efficacy of spindle inhibitors for the treatment of glioblastoma. Highlights* Resistance to non-microtubule spindle inhibitors limits their efficacy in glioblastoma and depends on STAT3. * Resistance goes hand in hand with development of therapy induced senescence (TIS). * Spindle inhibitor resistant glioblastomas consist of three cell subpopulations--proliferative, quiescent, and TIS--with proliferative cells sensitive and quiescent and TIS cells resistant. * TIS cells secrete TGF{beta}, which induces proliferative cells to become quiescent, thereby expanding the population of resistant cells in a spindle inhibitor resistant glioblastoma * Treatment with a STAT3 inhibitor kills TIS cells and restores sensitivity to spindle inhibitors.

cancer biology↗

bioRxiv2024

Stable, chronic in-vivo recordings from a fully wireless subdural-contained 65,536-electrode brain-computer interface device

Minimally invasive, high-bandwidth brain-computer-interface (BCI) devices can revolutionize human applications. With orders-of-magnitude improvements in volumetric efficiency over other BCI technologies, we developed a 50-m-thick, mechanically flexible micro-electrocorticography (ECoG) BCI, integrating a 256x256 array of electrodes, signal processing, data telemetry, and wireless powering on a single complementary metal-oxide-semiconductor (CMOS) substrate containing 65,536 recording channels, from which we can simultaneously record a selectable subset of up to 1024 channels at a given time. Fully implanted below the dura, our chip is wirelessly powered, communicating bi-directionally with an external relay station outside the body. We demonstrated chronic, reliable recordings for up to two weeks in pigs and up to two months in behaving non-human primates from somatosensory, motor, and visual cortices, decoding brain signals at high spatiotemporal resolution.

neuroscience↗

bioRxiv2024

PDGF-BB overexpression in p53 null oligodendrocyte progenitors increases H3K27me3 and induces transcriptional changes which favor proliferation

Proneural gliomas are brain tumors characterized by enrichment of oligodendrocyte progenitor cell (OPC) transcripts and genetic alterations. In this study we sought to identify transcriptional and epigenetic differences between OPCs with Trp53 deletion and PDGF-BB overexpression (BB-p53n), which form tumors when transplanted in mouse brains, and those carrying only p53 deletion (p53n), which do not. We used unbiased histone proteomics and RNA-seq analysis on these two genetically modified OPC populations and detected higher levels of H3K27me3 in BB-p53n compared to p53n OPCs. The BB-p53n OPC were characterized by higher levels of transcripts related to proliferation and lower levels of those related to differentiation. Pharmacological inhibition of histone H3K27 trimethylation in BB-p53n OPC reduced cell cycle transcripts and increased the expression of differentiation markers. These data suggest that PDGF-BB overexpression in p53 null OPC results in histone post-translational modifications and consequent transcriptional changes favoring proliferation while halting differentiation, thereby promoting the early stages of transformation.

neuroscience↗

bioRxiv2024

A pharmacological toolkit for human microglia identifies Topoisomerase I inhibitors as immunomodulators for Alzheimer's disease

AbstractWhile efforts to identify microglial subtypes have recently accelerated, the relation of transcriptomically defined states to function has been largely limited to in silico annotations. Here, we characterize a set of pharmacological compounds that have been proposed to polarize human microglia towards two distinct states - one enriched for AD and MS genes and another characterized by increased expression of antigen presentation genes. Using different model systems including HMC3 cells, iPSC-derived microglia and cerebral organoids, we characterize the effect of these compounds in mimicking human microglial subtypes in vitro. We show that the Topoisomerase I inhibitor Camptothecin induces a CD74high/MHChigh microglial subtype which is specialized in amyloid beta phagocytosis. Camptothecin suppressed amyloid toxicity and restored microglia back to their homeostatic state in a zebrafish amyloid model. Our work provides avenues to recapitulate human microglial subtypes in vitro, enabling functional characterization and providing a foundation for modulating human microglia in vivo.

neuroscience↗

bioRxiv2024

Glioma-Induced Alterations in Excitatory Neurons are Reversed by mTOR Inhibition

Gliomas are highly aggressive brain tumors characterized by poor prognosis and composed of diffusely infiltrating tumor cells that intermingle with non-neoplastic cells in the tumor microenvironment, including neurons. Neurons are increasingly appreciated as important reactive components of the glioma microenvironment, due to their role in causing hallmark glioma symptoms, such as cognitive deficits and seizures, as well as their potential ability to drive glioma progression. Separately, mTOR signaling has been shown to have pleiotropic effects in the brain tumor microenvironment, including regulation of neuronal hyperexcitability. However, the local cellular-level effects of mTOR inhibition on glioma-induced neuronal alterations are not well understood. Here we employed neuron-specific profiling of ribosome-bound mRNA via RiboTag, morphometric analysis of dendritic spines, and in vivo calcium imaging, along with pharmacological mTOR inhibition to investigate the impact of glioma burden and mTOR inhibition on these neuronal alterations. The RiboTag analysis of tumor-associated excitatory neurons showed a downregulation of transcripts encoding excitatory and inhibitory postsynaptic proteins and dendritic spine development, and an upregulation of transcripts encoding cytoskeletal proteins involved in dendritic spine turnover. Light and electron microscopy of tumor-associated excitatory neurons demonstrated marked decreases in dendritic spine density. In vivo two-photon calcium imaging in tumor-associated excitatory neurons revealed progressive alterations in neuronal activity, both at the population and single-neuron level, throughout tumor growth. This in vivo calcium imaging also revealed altered stimulus-evoked somatic calcium events, with changes in event rate, size, and temporal alignment to stimulus, which was most pronounced in neurons with high-tumor burden. A single acute dose of AZD8055, a combined mTORC1/2 inhibitor, reversed the glioma-induced alterations on the excitatory neurons, including the alterations in ribosome-bound transcripts, dendritic spine density, and stimulus evoked responses seen by calcium imaging. These results point to mTOR-driven pathological plasticity in neurons at the infiltrative margin of glioma - manifested by alterations in ribosome-bound mRNA, dendritic spine density, and stimulus-evoked neuronal activity. Collectively, our work identifies the pathological changes that tumor-associated excitatory neurons experience as both hyperlocal and reversible under the influence of mTOR inhibition, providing a foundation for developing therapies targeting neuronal signaling in glioma.

neuroscience↗

bioRxiv2023

Consensus scHPF Identifies Cell Type-Specific Drug Responses in Glioma by Integrating Large-Scale scRNA-seq

Single-cell transcriptomic analyses now frequently involve elaborate study designs including samples from multiple individuals, experimental conditions, perturbations, and batches from complex tissues. Dimensionality reduction is required to facilitate integration, interpretation, and statistical analysis. However, these datasets often include subtly different cellular subpopulations or state transitions, which are poorly described by clustering. We previously reported a Bayesian matrix factorization algorithm called single-cell hierarchical Poisson factorization (scHPF) that identifies gene co-expression patterns directly from single-cell RNA-seq (scRNA-seq) count matrices while accounting for transcript drop-out and noise. Here, we describe consensus scHPF, which analyzes scHPF models from multiple random initializations to identify the most robust gene signatures and automatically determine the number of factors for a given dataset. Consensus scHPF facilitates integration of complex datasets with highly multi-modal posterior distributions, resulting in factors that can be uniformly analyzed across individuals and conditions. To demonstrate the utility of consensus scHPF, we performed a meta-analysis of a large-scale scRNA-seq dataset from drug-treated, human glioma slice cultures generated from surgical specimens across three major cell types, 19 patients, 10 drug treatment conditions, and 52 samples. In addition to recapitulating previously reported cell type-specific drug responses from smaller studies, consensus scHPF identified disparate effects of the topoisomerase poisons etoposide and topotecan that are highly consistent with the distinct roles and expression patterns of their respective protein targets.

genomics↗