Biology subjects

Freeman, M. R.

Publications and source records attributed to Freeman, M. R..

At least 19 recordsLinked to original sources

bioRxiv2026

Large oncosomes reprogram bone marrow mesenchymal stem cells to establish a pre-metastatic niche

Metastatic progression depends on the systemic remodeling of distant tissues before tumor cell arrival, yet the cancer-derived signals that orchestrate this process remain poorly understood. Large oncosomes (LOs) are atypically large (>1 um), tumor-derived extracellular vesicles shed by invasive cancer cells. Here, we demonstrate that LOs function as systemic mediators of pre-metastatic niche formation by activating innate immune sensing in bone marrow mesenchymal stem cells (BM-MSCs). Systemic administration of prostate- and breast cancer-derived LOs to immunocompetent tumor-bearing mice did not affect primary tumor growth but increased metastatic burden. LOs induced a robust, dose-dependent interferon-driven inflammatory program, characterized by interferon-stimulated genes and neutrophil chemokines. Mechanistically, this response was driven by LO-associated nucleic acids activating convergent cytosolic DNA- and RNA-sensing pathways in recipient stromal cells. LO-conditioned BM-MSCs promoted the accumulation and polarization of neutrophils toward an immunosuppressive, polymorphonuclear myeloid-derived suppressor cell (PMN-MDSC) phenotype. In vivo, genetic suppression of LO shedding in tumor cells reduced PMN-MDSC accumulation in the bone marrow, which was reverted by systemic LO administration. Together, these findings establish LOs as specialized carriers of immunomodulatory signals that reprogram the bone marrow microenvironment to support metastatic colonization, identifying a previously unrecognized mechanism linking tumor vesiculation to immune remodeling at distant sites.

cancer biology↗

bioRxiv2026

All-in-one, Cas13d-based cell-specific gene knockdown system for zebrafish

Cell type-specific genetic manipulation is essential for dissecting neural circuits and glial biology in vivo; however, methods for achieving and validating cell-specific gene knockdown in zebrafish remain limited. Here, we introduce a CRISPR-Cas13d-based mRNA knockdown platform for zebrafish that leverages RNA targeting, rather than DNA editing, to enable efficient and flexible perturbation of gene function in defined cell types of interest. We engineered an all-in-one vector system for cell type-specific RfxCas13d expression and ubiquitous expression of crispr RNAs (crRNAs) and validated its utility across the major classes of CNS glia: astrocytes, oligodendrocytes, and microglia. Cas13d-mediated knockdown consistently achieved target mRNA depletion and produced heritable reproducible morphological and functional phenotypes, demonstrating the robustness of the approach. This approach complements permanent genome-editing methods such as CRISPR-Cas9 by providing a reliable, efficient, and scalable strategy for RNA-level perturbation. Our Cas13d toolkit expands the repertoire of zebrafish genetic technologies, offering a powerful resource for in vivo cell type-specific studies to uncover cell-autonomous mechanisms and possibility for cell type-specific genetic screens in development and disease.

neuroscience↗

bioRxiv2025

Comparison of QuPath and HALO platforms for analysis of the tumor microenvironment in prostate cancer

QuPath, an open-source digital pathology platform, has gained widespread use for image analysis in biomedical research since its release in 2016. However, its reproducibility and reliability compared to commercial software, such as HALO, requires further validation, particularly for multiplex immunofluorescence (mIF) analysis. In this study, we performed a direct comparison of QuPath and HALO using a mIF-stained prostate cancer tissue microarray (TMA) inclusive of 192 unique cores. We evaluated performance across three key analytical modules: immune cell phenotyping, tumor infiltration with immune cells, and nearest neighbor analysis. Furthermore, we integrated QuPath with CytoMap, an open-source spatial analysis tool, to perform unsupervised clustering of immune cell infiltration--a feature not available in HALO. Our results demonstrated high concordance between two platforms, with correlation coefficients exceeding 0.89 for immune cell density, distance and pattern of cell organization in tumor microenvironment (TME). A neighborhood analysis using CytoMap was further performed and provided a more detailed spatial analysis of immune cell distribution across different prostate cancer grades. A significant increase of CD103+ T cell infiltration into TME was observed in prostate cancer, which might be associated with the expression level of its ligand (E-cadherin) in the tumor region. In conclusion, our findings validate QuPath as a robust and reproducible alternative to commercial platforms for fluorescence-based digital pathology. By demonstrating QuPaths capability to perform high-quality quantitative analysis with additional flexibility for integration with external tools, our study underscores its potential for advancing tumor microenvironment research in translational oncology.

pathology↗

bioRxiv2025

Glia phagocytose neuronal sphingolipids to infiltrate developing synapses

The complex morphologies of mature neurons and glia emerge through profound rearrangements of cell membranes during development. Despite being integral components of these membranes, it is unclear whether lipids might actively sculpt these morphogenic processes. By analyzing lipid levels in the developing fruit fly brain, we discover dramatic increases in specific sphingolipids coinciding with neural circuit establishment. Disrupting this sphingolipid bolus via genetic perturbations of sphingolipid biosynthesis and catabolism leads to impaired glial autophagy. Remarkably, glia can obtain sphingolipid precursors needed for autophagy by phagocytosing neurons. These precursors are then converted into specific long-chain ceramide phosphoethanolamines (CPEs), invertebrate analogs of sphingomyelin. These lipids are essential for glia to arborize and infiltrate the brain, a critical step in circuit maturation that when disrupted leads to reduced synapse numbers. Taken together, our results demonstrate how spatiotemporal tuning of sphingolipid metabolism during development plays an instructive role in programming brain architecture. HighlightsO_LIBrain sphingolipids (SLs) remodel to very long-chain species during circuit maturation C_LIO_LIGlial autophagy requires de novo SL biosynthesis coordinated across neurons and glia C_LIO_LIGlia evade a biosynthetic blockade by phagolysosomal salvage of neuronal SLs C_LIO_LICeramide Phosphoethanolamine is critical for glial infiltration and synapse density C_LI

neuroscience↗

bioRxiv2024

Drosophila with-no-lysine and frayed Act in Parallel to dSarm to Drive Neurodegeneration Following dNmnat Depletion

How neurons to sense when they are terminally dysfunctional and activate neurodegeneration remains poorly defined. The pro-degenerative NAD+ hydrolase dSarm/SARM1 can act as a metabolic sensor by detecting pathological changes in NAD+/NMN and subsequently induce catastrophic axon degeneration. Here we show Drosophila with-no-lysine kinase (dWnk), which can directly sense Cl-, K+ and osmotic pressure, is required for neurodegeneration induced by depletion of the NAD+ biosynthetic enzyme dNmnat. dWnk functions in parallel to dSarm and acts through the downstream kinase Frayed to promote axon degeneration and neuronal cell death. dWnk and dSarm ultimately converge on the BTB-Back domain molecule Axundead (Axed) to execute neurodegeneration. Our work argues that neurons use direct sensors of both metabolism (dSarm/SARM1) and ionic/osmotic status (dWnk) to evaluate cellular health and, when dysfunctional, promote neurodegeneration though a common axon death signaling molecule, Axundead.

neuroscience↗

bioRxiv2024

Adrenergic signaling gates astrocyte responsiveness to neurotransmitters and control of neuronal activity

How astrocytes regulate neuronal circuits is a fundamental, unsolved question in neurobiology. Nevertheless, few studies have explored the rules that govern when astrocytes respond to different neurotransmitters in vivo and how they affect downstream circuit modulation. Here, we report an unexpected mechanism in Drosophila by which G-protein coupled adrenergic signaling in astrocytes can control, or "gate," their ability to respond to other neurotransmitters. Further, we show that manipulating this pathway potently regulates neuronal circuit activity and animal behavior. Finally, we demonstrate that this gating mechanism is conserved in mammalian astrocytes, arguing it is an ancient feature of astrocyte circuit function. Our work establishes a new mechanism by which astrocytes dynamically respond to and modulate neuronal activity in different brain regions and in different behavioral states.

neuroscience↗

bioRxiv2024

The Human Cytomegalovirus vGPCR UL33 is Essential for Efficient Lytic Replication in Epithelial Cells

Human cytomegalovirus (HCMV) is a {beta}-herpesvirus which is ubiquitous in the human population. HCMV has the largest genome of all known human herpesviruses, and thus encodes a large array of proteins that affect pathogenesis in different cell types. Given the large genome and the ability of HCMV to replicate in a range of cells, investigators have begun to identify viral proteins required for cell type-specific replication. There are four proteins encoded in the HCMV genome that are homologous to human G protein-coupled receptors (GPCRs); these viral-encoded GPCRs (vGPCRs) are UL33, UL78, US27, and US28. In the current study, we find that deletion of all four vGPCR genes from a clinical isolate of HCMV severely attenuates lytic replication in both primary human salivary gland epithelial cells, as well as ARPE-19 retinal epithelial cells as evidenced by significant decreases in immediate early gene expression and virus production. Deletion of UL33 from the HCMV genome also results in a failure to efficiently replicate in epithelial cells, and this defect is manifested by decreased levels of immediate early, early, and late gene expression, as well as reduced viral production. We find that similar to US28, UL33 constitutively activates Gq-dependent PLC-{beta} signaling to high levels in these epithelial cells. We also find that UL33 transcription is more complicated than originally believed, and there is the potential for the virus to utilize various 5 UTRs to create novel UL33 proteins that are all capable of constitutive Gq signaling. Taken together, these studies suggest that UL33 driven signaling is important for lytic HCMV replication in cells of epithelial origin.

microbiology↗

bioRxiv2024

Extracellular Vesicles heterogeneity through the lens of multiomics

Extracellular vesicles (EVs) are heterogenous in size, biogenesis, cargo and function. Beside small EVs, aggressive tumor cells release a population of particularly large EVs, namely large oncosomes (LO). This study provides the first resource of label-free quantitative proteomics of LO and small EVs obtained from distinct cancer cell types (prostate, breast, and glioma). This dataset was integrated with a SWATH Proteomic assay on LO, rigorously isolated from the plasma of patients with metastatic prostate cancer (PC). Proteins enriched in LO, which were identified also at the RNA level, and found in plasma LO significantly correlated with PC progression. Single EV RNA-Seq of the PC cell-derived LO confirmed some of the main findings from the bulk RNA-Seq, providing first evidence that single cell technologies can be successfully applied to EVs. This multiomics resource of cancer EVs can be leveraged for developing a multi-analyte approach for liquid biopsy.

cancer biology↗

bioRxiv2023

Epigenetic disruption of the RARγ complex impairs its function to bookmark AR enhancer interactions required for enzalutamide sensitivity in prostate cancer

The current study in prostate cancer (PCa) focused on the genomic mechanisms at the cross-roads of pro-differentiation signals and the emergence of lineage plasticity. We explored an understudied cistromic mechanism involving RAR{gamma}s ability to govern AR cistrome-transcriptome relationships, including those associated with more aggressive PCa features. The RAR{gamma} complex in PCa cell models was enriched for canonical cofactors, as well as proteins involved in RNA processing and bookmarking. Identifying the repertoire of miR-96 bound and regulated gene targets, including those recognition elements marked by m6A, revealed their significant enrichment in the RAR{gamma} complex. RAR{gamma} significantly enhanced the AR cistrome, particularly in active enhancers and super-enhancers, and overlapped with the binding of bookmarking factors. Furthermore, RAR{gamma} expression led to nucleosome-free chromatin enriched with H3K27ac, and significantly enhanced the AR cistrome in G2/M cells. RAR{gamma} functions also antagonized the transcriptional actions of the lineage master regulator ONECUT2. Similarly, gene programs regulated by either miR-96 or antagonized by RAR{gamma} were enriched in alternative lineages and more aggressive PCa phenotypes. Together these findings reveal an under-investigated role for RAR{gamma}, modulated by miR-96, to bookmark enhancer sites during mitosis. These sites are required by the AR to promote transcriptional competence, and emphasize luminal differentiation, while antagonizing ONECUT2.

cancer biology↗

bioRxiv2023

ONECUT2 Activates Diverse Resistance Drivers of Androgen Receptor-Independent Heterogeneity in Prostate Cancer

Significance StatementONECUT2 (OC2) is a master transcription factor that alters lineage identity by activating gene networks associated with both neuroendocrine prostate cancer and prostate adenocarcinoma. A small molecule inhibitor of OC2 represses the lineage plasticity program activated by enzalutamide, suggesting OC2 inhibition as a novel therapeutic strategy to prevent emergence of treatment-resistant variants. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=135 SRC="FIGDIR/small/560025v2_ufig1.gif" ALT="Figure 1"> View larger version (41K): org.highwire.dtl.DTLVardef@13278a4org.highwire.dtl.DTLVardef@8e82aeorg.highwire.dtl.DTLVardef@25a115org.highwire.dtl.DTLVardef@728eb4_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOGraphic AbstractC_FLOATNO C_FIG Androgen receptor-(AR-) indifference is a mechanism of resistance to hormonal therapy in prostate cancer (PC). Here we demonstrate that the HOX/CUT transcription factor ONECUT2 (OC2) activates resistance through multiple drivers associated with adenocarcinoma, stem-like and neuroendocrine (NE) variants. Direct OC2 targets include the glucocorticoid receptor and the NE splicing factor SRRM4, among others. OC2 regulates gene expression by promoter binding, enhancement of chromatin accessibility, and formation of novel super-enhancers. OC2 also activates glucuronidation genes that irreversibly disable androgen, thereby evoking phenotypic heterogeneity indirectly by hormone depletion. Pharmacologic inhibition of OC2 suppresses lineage plasticity reprogramming induced by the AR signaling inhibitor enzalutamide. These results demonstrate that OC2 activation promotes a range of drug resistance mechanisms associated with treatment-emergent lineage variation in PC. Our findings support enhanced efforts to therapeutically target this protein as a means of suppressing treatment-resistant disease.

cancer biology↗

bioRxiv2023

The homeodomain drives favorable DNA binding energetics of prostate cancer target ONECUT2

The ONECUT transcription factors feature a CUT and a homeodomain, evolutionarily conserved elements that bind DNA cooperatively, but the process remains mechanistically enigmatic. Using an integrative DNA binding analysis of ONECUT2, a driver of aggressive prostate cancer, we show that the homeodomain energetically stabilizes the ONECUT2-DNA complex through allosteric modulation of CUT. Further, evolutionarily conserved base-interactions in both the CUT and homeodomain are necessary for the favorable thermodynamics. We have identified a novel arginine pair unique to the ONECUT family homeodomain that can adapt to DNA sequence variations. Base interactions in general, including by this arginine pair, are critical for optimal DNA binding and transcription in a prostate cancer model. These findings provide fundamental insights into DNA binding by CUT-homeodomain proteins with potential therapeutic implications. One-Sentence SummaryBase-specific interactions regulate homeodomain-mediated stabilization of DNA binding by the ONECUT2 transcription factor.

biophysics↗

bioRxiv2022

ONECUT2 restricts Microfold cell numbers in the small intestine; a multi-omics study

Microfold (M) cells reside in the intestinal epithelium of Peyers patches. Their unique ability to take up and transport antigens from the intestinal lumen to the underlying lymphoid tissue is key in the regulation of the gut-associated immune response. Here, we applied a (single-cell) multi-omics approach to investigate the molecular mechanisms that drive M cell differentiation in mouse small intestinal organoids. We generated a comprehensive profile of chromatin accessibility changes and transcription factor dynamics during in vitro M cell differentiation, allowing us to uncover numerous cell type-specific regulatory elements and associated transcription factors. Single-cell RNA sequencing resulted in the identification of an M cell precursor population. Our new computational tool SCEPIA determined that these precursor cells were characterized by high expression of and motif activity for the transcription factor ONECUT2. Subsequent perturbation experiments revealed that ONECUT2 acts downstream of the RANK/RANKL signalling to support Enterocyte differentiation and restrict M cell lineage specification in vitro and in vivo, thereby regulating mucosal immunity. This study provides a useful blueprint for future investigations of cell fate switches in the intestinal epithelium.

molecular biology↗

bioRxiv2021

Discoidin domain receptor regulates ensheathment, survival, and caliber of peripheral axons

Invertebrate axons and small caliber axons in mammalian peripheral nerves are unmyelinated but still ensheathed by glia. How this type of ensheathment is controlled and its roles in supporting neuronal function remain unclear. Here we use Drosophila wrapping glia, which ensheathe peripheral axons to study the function and development of non-myelinating ensheathment. We developed a new SplitGal4 intersectional driver to target wrapping glia for genetic ablation and found that loss of wrapping glia severely impaired larval locomotor behavior. We performed an in vivo RNAi screen in Drosophila to identify glial genes required for axon ensheathment during development and identified the conserved receptor tyrosine kinase Discoidin domain receptor (Ddr). In larval peripheral nerves, loss of Ddr resulted in severely reduced ensheathment of axons. We found a strong dominant genetic interaction between Ddr and the fly type XV/XVIII collagen Multiplexin (Mp), suggesting Ddr functions a collagen receptor to drive wrapping of axons during development. Surprisingly, despite severe impairment of ensheathment, the residual wrapping in Ddr mutants was sufficient to support basic circuit function during larval stages. In adult nerves, loss of Ddr from glia decreased long-term survival of sensory neurons and significantly reduced axon caliber in an identifiable neuron without overtly affecting ensheathment. Our data establish a crucial role for non-myelinating glia in peripheral nerve development and function across the lifespan, and identify Ddr as a key regulator of axon-glia interactions during ensheathment and nerve growth.

neuroscience↗

bioRxiv2021

Astrocytic GABA Transporter controls sleep by modulating GABAergic signaling in Drosophila circadian neurons

A precise balance between sleep and wakefulness is essential to sustain a good quality of life and optimal brain function. GABA is known to play a key and conserved role in sleep control, and GABAergic tone should therefore be tightly controlled in sleep circuits. Here we examined the role of the astrocytic GABA transporter (GAT) in sleep regulation using Drosophila melanogaster. We found that a hypomorphic gat mutation (gat33-1) increased sleep amount, decreased sleep latency, and increased sleep consolidation. Interestingly, sleep defects were suppressed when gat33-1 was combined with a mutation disrupting wide-awake (wake), a gene that regulates the cell-surface levels of the GABAA receptor Resistance to Dieldrin (RDL) in the wake-promoting large ventral lateral neurons (l-LNvs). Moreover, RNAi knockdown of rdl and its modulator dnlg4 in these circadian neurons also suppressed gat33-1 sleep phenotypes. Brain immunohistochemistry showed that GAT-expressing astrocytes were located near RDL-positive l-LNvs cell bodies and dendritic processes. We conclude that astrocytic GAT decreases GABAergic tone and RDL activation in arousal promoting LNvs, thus determining proper sleep amount and quality in Drosophila

neuroscience↗

bioRxiv2020

RIPK2 Stabilizes c-Myc and is an Actionable Target for Inhibiting Prostate Cancer Metastasis

Despite advances in diagnosis and treatment, metastatic prostate cancer remains incurable and is associated with high mortality rates. Thus, novel actionable drug targets are urgently needed for therapeutic interventions in advanced prostate cancer. Here we report receptor-interacting protein kinase 2 (RIPK2) as an actionable drug target for suppressing prostate cancer metastasis. RIPK2 is frequently amplified in lethal prostate cancers and its overexpression is associated with disease progression and aggressiveness. Genetic and pharmacological inhibition of RIPK2 significantly suppressed prostate cancer progression in vitro and metastasis in vivo. Multi-level proteomic analysis revealed that RIPK2 strongly regulates c-Myc protein stability and activity, largely by activating the MKK7/JNK/c-Myc phosphorylation pathway--a novel, non-canonical RIPK2 signaling pathway. Targeting RIPK2 inhibits this phosphorylation pathway, and thus promotes the degradation of c-Myc--a potent oncoprotein for which no drugs have been approved for clinical use yet. These results support targeting RIPK2 for personalized therapy in prostate cancer patients towards improving survival.

cancer biology↗

bioRxiv2020

Astrocytes close a critical period of motor circuit plasticity

Critical periods - brief intervals where neural circuits can be modified by sensory input - are necessary for proper neural circuit assembly. Extended critical periods are associated with neurodevelopmental disorders, including schizophrenia and autism; however, the mechanisms that ensure timely critical period closure remain unknown. Here, we define the extent of a critical period in the developing Drosophila motor circuit, and identify astrocytes as essential for proper critical period termination. During the critical period, decreased activity produces larger motor dendrites with fewer inhibitory inputs; conversely, increased motor neuron activity produces smaller motor dendrites with fewer excitatory inputs. Importantly, activity has little effect on dendrite morphology after critical period closure. Astrocytes invade the neuropil just prior to critical period closure, and astrocyte ablation prolongs the critical period. Finally, we use a genetic screen to identify astrocyte-motor neuron signaling pathways that close the critical period, including Neuroligin-Neurexin signaling. Reduced signaling destabilizes dendritic microtubules, increases dendrite dynamicity, and impairs locomotor behavior, underscoring the importance of critical period closure. Previous work defines astroglia as regulators of plasticity at individual synapses; here, we show that astrocytes also regulate large-scale structural plasticity to motor dendrite, and thus, circuit architecture to ensure proper locomotor behavior.

neuroscience↗

bioRxiv2019

TrpML-mediated astrocyte microdomain Ca2+ transients regulate astrocyte-tracheal interactions in CNS

Astrocytes exhibit spatially-restricted near-membrane microdomain Ca2+ transients in their fine processes. How these transients are generated and regulate brain function in vivo remains unclear. Here we show that Drosophila astrocytes exhibit spontaneous, activity-independent microdomain Ca2+ transients in their fine processes. Astrocyte microdomain Ca2+ transients are mediated by the TRP channel TrpML, stimulated by reactive oxygen species (ROS), and can be enhanced in frequency by the neurotransmitter tyramine via the TyrRII receptor. Interestingly, many astrocyte microdomain Ca2+ transients are closely associated with tracheal elements, which dynamically extend filopodia throughout the central nervous system (CNS) to deliver O2 and regulate gas exchange. Many astrocyte microdomain Ca2+ transients are spatio-temporally correlated with the initiation of tracheal filopodial retraction. Loss of TrpML leads to increased tracheal filopodial numbers, growth, and increased CNS ROS. We propose that local ROS production can activate astrocyte microdomain Ca2+ transients through TrpML, and that a subset of these microdomain transients promotes tracheal filopodial retraction and in turn modulate CNS gas exchange.

neuroscience↗

bioRxiv2019

Comprehensive palmitoyl-proteomic analysis identifies distinct protein signatures for large and small cancer-derived extracellular vesicles

Extracellular vesicles (EVs) are membrane-enclosed particles that play an important role in cancer progression and have emerged as a promising source of circulating biomarkers. Protein S-acylation, also known as palmitoylation, has been proposed as a post-translational mechanism that modulates the dynamics of EV biogenesis and protein cargo sorting. However, technical challenges have limited large-scale profiling of the whole palmitoyl-proteins of EVs. We successfully employed a novel approach that combines low-background acyl-biotinyl exchange (LB-ABE) with label-free proteomics to analyze the palmitoyl proteome of large EVs (L-EVs) and small EVs (S-EVs) from prostate cancer cells. Here we report the first palmitoyl-protein signature of EVs, and demonstrate that L- and S-EVs harbor proteins associated with distinct biological processes and subcellular origin. We identified STEAP1, STEAP2, and ABBC4 as prostate cancer-specific palmitoyl proteins enriched in both EV populations in comparison with the originating cell lines. Importantly, the presence of the above proteins in EVs was significantly reduced upon inhibition of palmitoylation in the producing cells. These results suggest that palmitoylation may be involved in the differential sorting of proteins to distinct EV populations and allow for better detection of disease biomarkers.

cancer biology↗