Biology subjects

Dunn, G. P.

Publications and source records attributed to Dunn, G. P..

11 recordsLinked to original sources

bioRxiv2026

Integrated spatial and single-cell transcriptomic analysis of aggressive glioblastoma growth dynamics.

Glioblastoma (GBM) develops within a complex tumor ecosystem whose temporal dynamics remain poorly understood. Here, we performed longitudinal single-cell RNA sequencing and spatial transcriptomics across multiple timepoints in two widely used murine GBM models - CT2A and GL261 - which differ markedly in aggressiveness and response to immune checkpoint blockade. Tumor cell transcriptomes revealed model-specific programs: CT2A cells progressively upregulated epithelial-mesenchymal transition (EMT), non-classical MHC Class I, and progressively, hypoxia response pathways, resembling the human mesenchymal GBM cell state, while GL261 cells exhibited MHC Class II expression and developmental signatures resembling oligodendrocyte progenitor and astrocytic states. Ligand-receptor interaction analyses identified thrombospondins (Thbs1, Thbs2) and osteopontin (Spp1) as CT2A-specific tumor ligands mediating tumorigenic interactions with immune cells, with downstream targets enriched for EMT and TGF-{beta} pathways. Conversely, the GL261 model presented a differential potential to engage neuronal and perivascular guidance networks, with Glutamate and L1 cell adhesion molecule (L1cam) as lead signaling partners. The CT2A immune compartment exhibited progressive microglia-to-macrophage phenotypic conversion, enhanced macrophage infiltration driven by Spp1, and elevated T cell exhaustion, while GL261 maintained a distinct adaptive immune communication hub via MHC class II-CD4 signaling. Elevated THBS1, THBS2, and SPP1 expression correlated with poor survival in human GBM datasets. Together, these findings reveal divergent tumor-immune ecosystems in CT2A and GL261 that recapitulate distinct aspects of human GBM, with implications for therapeutic targeting.

cancer biology↗

bioRxiv2025

Mapping CD4+ T Cell Landscapes in Glioblastoma Reveals Effectors and Bystanders

The tumor immune microenvironment (TME) has been demonstrated to significantly shape glioblastoma (GBM) progression and therapeutic response, yet the role of CD4+ T cells remains incompletely defined. Here, by integrating single-cell RNA, surface-protein, and TCR profiling of 39,788 T cells from 16 high-grade gliomas with five matched blood samples, we mapped 23,550 CD4+ T cells across 11 states and revealed predominance of CD4+ T cells in primary, but not recurrent, tumors, alongside pronounced tumor-blood discordance. Clonal analyses revealed expansion of cytotoxic effector CD4+ T cells within tumors and TEMRA CD4+ T cells in blood. A small set of dominant clonotypes (0.2% of unique TCR{beta} sequences) accounted for >6% of all CD4+ T cells across both compartments and were shared across different transcriptional subsets, suggesting diverse transcriptional development stemming from a shared progenitor. In contrast, virally annotated clonotypes were broadly dispersed and largely unexpanded, consistent with bystander populations. Collectively, we investigated the cellular and clonal architecture of human CD4+ T cells in GBM and highlight the contrast between PBMC and TIL compartments.

immunology↗

bioRxiv2025

Single-Cell Analysis of Meningiomas Reveals Mutation-Associated Tumor and Immune Cell Gene Expression Programs

Genomic and epigenetic profiling, particularly DNA methylation analysis, have refined the molecular classification of meningiomas and revealed marked intratumoral heterogeneity. To further characterize heterogeneity within and between patients, we analyzed meningiomas using single-cell RNA sequencing (n=11), whole-exome sequencing (n=9), and spatial transcriptomics (n=3). Single-cell analysis revealed six transcriptionally distinct tumor cell states that corresponded to unique biological processes. Integration of the single-cell data with published exome and bulk RNA sequencing data from a large cohort revealed significant associations among somatic variants, tumor cell states, and immunological signatures. Notably, NF2-altered tumors were enriched for an epithelial-to-mesenchymal transition (EMT) cell state and immune cells, whereas NF2-intact tumors were enriched for a sterol metabolism cell state. Spatial transcriptomic analysis confirmed co-localization of immune cells and EMT tumor cells. Comparisons with immune cells from other brain tumors and peripheral tissues highlighted immunological cell states specific to meningioma. Collectively, these findings refine our genetic and molecular understanding of meningioma heterogeneity and underscore the link between genotype and molecular phenotype.

cancer biology↗

bioRxiv2025

Mapping the spatial architecture of glioblastoma from core to edge delineates niche-specific tumor cell states and intercellular interactions

Treatment resistance in glioblastoma (GBM) is largely driven by the extensive multi-level heterogeneity that typifies this disease. Despite significant progress toward elucidating GBMs genomic and transcriptional heterogeneity, a critical knowledge gap remains in defining this heterogeneity at the spatial level. To address this, we employed spatial transcriptomics to map the architecture of the GBM ecosystem. This revealed tumor cell states that are jointly defined by gene expression and spatial localization, and multicellular niches whose composition varies along the tumor core-edge axis. Ligand-receptor interaction analysis uncovered a complex network of intercellular communication, including niche- and region-specific interactions. Finally, we found that CD8 positive GZMK positive T cells colocalize with LYVE1 positive CD163 positive myeloid cells in vascular regions, suggesting a potential mechanism for immune evasion. These findings provide novel insights into the GBM tumor microenvironment, highlighting previously unrecognized patterns of spatial organization and intercellular interactions, and novel therapeutic avenues to disrupt tumor-promoting interactions and overcome immune resistance.

cancer biology↗

bioRxiv2025

The producer cell type of HSV-1 alters the proteomic contents and infectious capacity of virions

The cell that a virus replicates in i.e., the producer cell, can alter the macromolecular composition and infectious capacity of the virions that are produced. Herpes Simplex virus type 1 (HSV-1) primarily infects keratinocytes of the epidermis or oral mucosa prior to establishing latency in neurons of the peripheral nervous system, where the virus can persist for the lifetime of the host. Many cell lines that are used to amplify HSV-1 are derived from species and tissue types that are less physiologically relevant to HSV-1 disease. To understand if the producer cell type influences HSV-1 infection, we tested the infectivity of HSV-1 derived from immortalized African green monkey kidney cells (vero), immortalized human keratinocytes (HaCaT), and primary human foreskin fibroblasts (HFF-1). We observed that the producer cell type alters the capacity of HSV-1 to produce viral proteins and infectious virions from infected cells and susceptibility to inhibition of replication by interferon treatment. HaCaT-derived HSV-1 consistently exhibited enhanced replication over HFF-1 or vero-derived virus. To determine if the producer cell type changes the protein composition of virions, we performed an untargeted LC/MS-MS analysis of virions purified from each cell line. Comparison of virion associated proteins revealed quantitative differences in composition of both cellular and viral proteins including ICP0, pUL24 and pUL42. These results highlight the influence that the producer cell-type has on HSV-1 infection outcomes and suggest that cell type specific factors can alter HSV-1 and impact viral replication. ImportanceApproximately 67% of the human population harbors HSV-1 infection. To study HSV-1 infection, laboratories utilize several different cell lines to propagate HSV-1 for downstream experiments. The type of cell used to produce a virus, i.e. the producer cell type, can alter the macromolecular composition, immunogenicity, and infectivity of the virions that are produced across several virus families. We found that the producer cell type of HSV-1 alters virion infectivity and virion protein composition. Therefore, the producer cell type may have implications in the spread of HSV-1 and subsequent disease outcomes in humans. Our results also raise concerns about how the use of different ceil types to propagate HSV-1 may alter the outcome, interpretation, and reproducibility of experimental results.

microbiology↗

bioRxiv2025

Intratumoral gene delivery of 4-1BBL boosts IL-12-triggered anti-glioblastoma immunity

The standard of care in high-grade gliomas has remained unchanged in the past 20 years. Efforts to replicate effective immunotherapies in non-cranial tumors have led to only modest therapeutical improvements in glioblastoma (GB). Here, we demonstrate that intratumoral administration of recombinant interleukin-12 (rIL-12) promotes local cytotoxic CD8POS T cell accumulation and conversion into an effector-like state, resulting in a dose-dependent survival benefit in preclinical GB mouse models. This tumor-reactive CD8 T cell response is further supported by intratumoral rIL-12-sensing dendritic cells (DCs) and is accompanied by the co-stimulatory receptor 4-1BB expression on both cell types. Given that DCs and CD8POS T cells are functionally suppressed in the tumor microenvironments of de novo and recurrent glioma patients, we tested whether anti-tumor response at the rIL-12-inflamed tumor site could be enhanced with 4-1BBL, the ligand of 4-1BB. 4-1BBL was delivered using an adeno-associated virus (AAV) vector targeting GFAP-expressing cells and resulted in prolonged survival of rIL-12 treated GB-bearing mice. This study establishes that tumor antigen-specific CD8 T cell activity can be directed using an AAV-vector-mediated gene therapy approach, effectively enhancing anti-GB immunity.

cancer biology↗

bioRxiv2023

Glioblastoma-infiltrating CD8+ T cells are predominantly a clonally expanded GZMK+ effector population

Recent clinical trials have highlighted the limited efficacy of T cell-based immunotherapy in patients with glioblastoma (GBM). To better understand the characteristics of tumor-infiltrating lymphocytes (TIL) in GBM, we performed cellular indexing of transcriptomes and epitopes by sequencing (CITE-seq) and single-cell RNA sequencing (scRNA-seq) with paired V(D)J sequencing, respectively, on TIL from two cohorts of patients totaling 15 patients with high grade glioma, including GBM or astrocytoma, IDH mutant, grade 4 (G4A). Analysis of the CD8+ TIL landscape reveals an enrichment of clonally expanded GZMK+ effector T cells in the tumor compared to matched blood, which was validated at the protein level. Furthermore, integration with other cancer types highlights the lack of a canonically exhausted CD8+ T cell population in GBM TIL. These data suggest that GZMK+ effector T cells represent an important T cell subset within the GBM microenvironment and which may harbor potential therapeutic implications. SignificanceIn order to understand the limited efficacy of immune checkpoint blockade in GBM, we endeavor to understand the TIL landscape through a multi-omics approach. In this study, by highlighting the enrichment of GZMK+ effector T cells and lack of exhausted T cells, we provide a new potential mechanism of resistance to immunotherapy in GBM.

immunology↗

bioRxiv2023

A dual-fluorescent recombinant for live observation of Herpes simplex-type 1 infection outcomes.

Critical stages of lytic Herpes simplex type 1 (HSV-1) replication are marked by the sequential expression of immediate early (IE) to early (E), then late (L) viral genes. HSV-1 also persists in neuronal tissues via a non-replicative, transcriptionally repressed infection called latency. Understanding the regulation of lytic and latent transcriptional profiles provides focused insight into HSV-1 infection and disease. We sought a fluorescence-based approach to observe temporal progression of HSV-1 infection at the single-cell level. We constructed and characterized a novel HSV-1 recombinant that reports IE and L gene expression by fluorescent protein detection. The dual-reporter HSV-1 visualizes IE gene expression by a CMV promotor-driven YFP, and L gene expression by an endogenous mCherry-VP26 fusion. We confirmed that viral gene expression, replication and spread of infection in epithelial cells is not altered by the incorporation of the fluorescent reporters. Interference with viral DNA polymerase activity abolishes VP26-mCherry detection late in HSV-1 infection, visually reporting the failure to complete viral replication. Viral replication in primary neurons is not altered, but retrograde-directed inoculation of the dual-reporter HSV-1 exhibits a modest reduction in titer, compared to unlabeled HSV-1. Low-dose axonal inoculation in the presence of small molecule modulation of neuronal signaling results in divergent outcomes of YFP and mCherry detection, suggesting different states of latent and lytic replication. Rigorous characterization of this dual-reporter HSV-1 recombinant has demonstrated the utility of temporal observation of HSV-1 replication in live cells and the potential for further insight into the dynamics of infection. ImportanceHerpes simplex virus-type 1 (HSV-1) is a prevalent human pathogen that infects approximately 67% of the global population. HSV-1 invades the peripheral nervous system, where latent HSV-1 infection persists within the host for life. Immunological evasion, viral persistence, and herpetic pathologies are determined by regulation of HSV-1 gene expression. Studying HSV-1 gene expression during neuronal infection is challenging but essential for the development of antiviral therapeutics and interventions. We constructed and characterized a dual-fluorescent HSV-1 recombinant that enables visualization of IE and L gene expression. The recombinant HSV-1 is used to observe the progression and outcome of infection. We demonstrate that drug treatments targeting cellular pathways can manipulate latent HSV-1 infection in neurons to achieve divergent outcomes of infection.

microbiology↗

bioRxiv2021

The conventional dendritic cell 1 subset primes CD8+ T cells and traffics tumor antigen to drive anti-tumor immunity in the brain

The central nervous system (CNS) antigen presenting cell (APC) which primes anti-tumor CD8+ T cell responses remains undefined. Elsewhere, the conventional dendritic cell 1 (cDC1) performs this role. However, steady-state brain cDC1 are rare; cDC localize to choroid plexus and dura. Using preclinical glioblastoma models and cDC1-deficient mice, we explored the role of cDC1 in CNS anti-tumor immunity. We determined that cDC1 mediate checkpoint blockade-induced survival benefit and prime neoantigen-specific CD8+ T cells against brain tumors. We observed that cDC, including cDC1, isolated from the tumor, the dura, and the CNS-draining cervical lymph nodes harbored a traceable fluorescent tumor-antigen. In patient samples, we observed several APC subsets (including the CD141+ cDC1-equivalent) infiltrating glioblastomas, meningiomas, and dura. In these same subsets, we identified a tumor-specific fluorescent metabolite of 5- aminolevulinic acid, which labels tumor cells during fluorescence-guided glioblastoma resection. Together, these data elucidate the specialized behavior of cDC1 and suggest cDC1 play a significant role in CNS anti-tumor immunity. One Sentence SummaryCNS cDC1 engage in previously undefined behavior to establish immune responses against brain tumors.

immunology↗

bioRxiv2020

Accurate neoantigen prediction depends on mutation position relative to patient allele-specific MHC anchor location

Neoantigens are novel peptide sequences resulting from sources such as somatic mutations in tumors. Upon loading onto major histocompatibility complex (MHC) molecules, they can trigger recognition by T cells. Accurate neoantigen identification is thus critical for both designing cancer vaccines and predicting response to immunotherapies. Neoantigen identification and prioritization relies on correctly predicting whether the presenting peptide sequence can successfully induce an immune response. As the majority of somatic mutations are single nucleotide variants, changes between wildtype and mutated peptides are typically subtle and require cautious interpretation. A potentially underappreciated variable in neoantigen-prediction pipelines is the mutation position within the peptide relative to its anchor positions for the patients specific MHC molecules. While a subset of peptide positions are presented to the T-cell receptor for recognition, others are responsible for anchoring to the MHC, making these positional considerations critical for predicting T-cell responses. We computationally predicted high probability anchor positions for different peptide lengths for 328 common HLA alleles and identified unique anchoring patterns among them. Analysis of 923 tumor samples shows that 6-38% of neoantigen candidates are potentially misclassified and can be rescued using allelespecific knowledge of anchor positions. A subset of anchor results were orthogonally validated using protein crystallography structures. Representative anchor trends were experimentally validated using peptide-MHC stability assays and competition binding assays. By incorporating our anchor prediction results into neoantigen prediction pipelines, we hope to formalize, streamline and improve the identification process for relevant clinical studies. One Sentence SummaryNeoantigen prediction accuracy is significantly influenced by the mutation position within the neoantigen and its relative position to the patients allele-specific MHC anchor locations.

bioinformatics↗

bioRxiv2017

Temporal and Clonal Progression in a Pediatric Ependymoma Patient Through Multiple Treatments

BackgroundMultiple recurrences after complete resection and irradiation of supratentorial ependymoma are common and frequently result in patient death. However, the molecular basis for treatment resistance, the impact that radiation and other adjuvant therapies have in promoting recurrence, and the use of this information to rationally design effective approaches to treat recurrent ependymoma are unknown. Due to the rarity of these tumors and the even less likely banking of multiple recurrent samples from the same patient, we initiated a study to characterize the evolution of a single patients ependymoma in response to therapy.\n\nMethods and FindingsA combination of high depth, whole genome and exome-based DNA sequencing of germline and tumor specimens, RNA sequencing of tumor specimens, and advanced computational analyses were employed to reconstruct the natural history of a supratentorial ependymoma case in which there were four local recurrences. The findings reveal the extent to which treatment with radiation and chemotherapies resulted in the diversification of the tumor subclonal architecture and shaped the neo-antigen landscape, and provide new insights into possible molecular mechanisms of oncogenesis, treatment response and recurrence.\n\nConclusionsAlthough the recurrent tumors we studied were clearly shaped by therapy, the founding clone was never eradicated by any treatment. We conclude that DNA and RNA sequencing may provide critical prognostic indicators to identify ependymoma patients that should be observed, rather than irradiated, post gross total resection.

cancer biology↗