Biology subjects

Furnari, J.

Publications and source records attributed to Furnari, J..

6 recordsLinked to original sources

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

Disrupted O-GalNAc glycosylation as a mechanism and biomarker of SLC35A2-associated epilepsy

Rare germline and somatic variants in SLC35A2 cause a spectrum of severe glycosylation disorders that commonly present with epilepsy. SLC35A2 encodes the Golgi transporter for UDP-galactose, but how its deficiency leads to severe neurodevelopmental disorders is unknown. Using a mouse model deficient for Slc35a2 in the forebrain, we identified a specific defect in O-GalNAc glycan synthesis, while other galactose-containing glycoconjugates remained intact. O-GalNAc glycans were absent from their normal location within neuronal tracts of the corpus callosum, and truncated precursors accumulated in the cortex on critical extracellular matrix molecules. Cultured primary neurons lacking Slc35a2 showed impaired development, hyperexcitability, and impaired O-GalNAc glycosylation. Finally, human brain tissue from cases of SLC35A2-associated intractable epilepsy displayed a strong correlation between variant burden and truncated O-GalNAc glycans. These findings provide a mechanistic link between genetic causes of SLC35A2-associated epilepsy and protein O-glycosylation that can be targeted for biomarker and therapeutic development.

neuroscience↗

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↗

bioRxiv2023

Scalable co-sequencing of RNA and DNA from individual nuclei

The ideal technology for directly investigating the relationship between genotype and phenotype would analyze both RNA and DNA genome-wide and with single-cell resolution. However, existing tools lack the throughput required for comprehensive analysis of complex tumors and tissues. We introduce a highly scalable method for jointly profiling DNA and expression following nucleosome depletion (DEFND-seq). In DEFND-seq, nuclei are nucleosome-depleted, tagmented, and separated into individual droplets for mRNA and genomic DNA barcoding. Once nuclei have been depleted of nucleosomes, subsequent steps can be performed using the widely available 10x Genomics droplet microfluidic technology and commercial kits without experimental modification. We demonstrate the production of high-complexity mRNA and gDNA sequencing libraries from thousands of individual nuclei from both cell lines and archived surgical specimens for associating gene expression phenotypes with both copy number and single nucleotide variants.

genomics↗

bioRxiv2022

Single-Cell Analysis of 5-ALA Intraoperative Labeling Specificity for Glioblastoma

Glioblastoma (GBM) is the most common and aggressive malignant primary brain tumor, and surgical resection is a key part of the standard-of-care. In fluorescence-guided surgery (FGS), fluorophores are used to differentiate tumor tissue from surrounding normal brain. The heme synthesis pathway converts 5-aminolevulinic acid (5-ALA), a fluorogenic substrate used for FGS, to fluorescent protoporphyrin IX (PpIX). The resulting fluorescence is thought to be specific to transformed glioma cells, but this specificity has not been examined at single-cell level. We performed paired single-cell imaging and RNA sequencing of individual cells (SCOPE-seq2) on human GBM surgical specimens with visible PpIX fluorescence from patients who received 5-ALA prior to surgery. SCOPE-seq2 allows us to simultaneously image PpIX fluorescence and unambiguously identify transformed glioma cells from single-cell RNA-seq (scRNA-seq). We observed that 5-ALA treatment results in labeling that is not specific to transformed tumor cells. In cell culture, we further demonstrated that untransformed cells can be labeled by 5-ALA directly or by PpIX secreted from surrounding transformed cells. In acute slice cultures from mouse glioma models, we showed that 5-ALA preferentially labels GBM tumor tissue over non-neoplastic brain tissue, and that this contrast is not due to blood-brain-barrier disruption. Taken together, our findings support the use of 5-ALA as an indicator of GBM tissue, but not as a specific marker of transformed glioma cells.

cancer biology↗

bioRxiv2021

Re-convolving the compositional landscape of primary and recurrent glioblastoma using single nucleus RNA sequencing

Glioblastoma (GBM) is an aggressive diffusely infiltrating neoplasm that spreads beyond surgical resection margins, where it intermingles with non-neoplastic brain cells. This complex microenvironment harboring infiltrating glioma and non-neoplastic brain cells is the origin of tumor recurrence. Thus, understanding the cellular and molecular features of the glioma microenvironment is therapeutically and prognostically important. We used single-nucleus RNA sequencing (snRNAseq) to determine the cellular composition and transcriptional states in primary and recurrent glioma and identified three compositional tissue-states defined by the observed patterns of cohabitation between neoplastic and non-neoplastic brain cells. These comprise states enriched in A) neurons and non-neoplastic glia, B) reactive astrocytes and inflammatory cells, and C) proliferating tumor cells. The tissue states also showed distinct associations with the different transcriptional states of GBM cells. Spatial transcriptomics revealed that the cell-types/transcriptional-states associated with each tissue state colocalize in space. Tissue states are clinically significant because they correlate with radiographic, histopathologic, and prognostic features. Importantly, we found that our compositionally-defined tissue states are enriched in distinct metabolic pathways. One such pathway is fatty acid biosynthesis, which was enriched in tissue state B - a state enriched in recurrent glioblastoma and associated with shorter overall survival- and composed of astrocyte-like/mesenchymal glioma cells, reactive astrocytes, and monocyte-like myeloid cells. We showed that treating acute slices of GBM with a fatty acid synthesis inhibitor is sufficient to deplete the transcriptional signature of tissue state B. Our findings define a novel compositional approach to analyze glioma-infiltrated tissue which allows us to discover prognostic and targetable features, paving the way to new mechanistic and therapeutic discoveries.

pathology↗