Multiple Mouse Research Articles

The interaction of tPA with NMDAR1 drives neuroinflammation and neurodegeneration in α-synuclein-mediated neurotoxicity

The thrombolytic protease tissue plasminogen activator (tPA) is expressed in the CNS, where it regulates diverse functions including neuronal plasticity, neuroinflammation, and blood-brain-barrier integrity. However, its role in different brain regions such as the substantia nigra (SN) is largely unexplored. In this study, we characterize tPA expression, activity, and localization in the SN using a combination of retrograde tracing and β-galactosidase tPA reporter mice. We further investigate tPA’s potential role in SN pathology in an α-synuclein mouse model of Parkinson’s disease (PD). To characterize the mechanism of tPA action in α-synuclein-mediated pathology in the SN and to identify possible therapeutic pathways, we performed RNA-seq analysis of the SN and used multiple transgenic mouse models. These included tPA deficient mice and two newly developed transgenic mice, a knock-in model expressing endogenous levels of proteolytically inactive tPA (tPA Ala-KI) and a second model overexpressing proteolytically inactive tPA (tPA Ala-BAC). Our findings show that striatal GABAergic neurons send tPA+ projections to dopaminergic (DA)-neurons in the SN and that tPA is released from SN-derived synaptosomes upon stimulation. We also found that tPA levels in the SN increased following α-synuclein overexpression. Importantly, tPA deficiency protects DA-neurons from degeneration, prevents behavioral deficits, and reduces microglia activation and T-cell infiltration induced by α-synuclein overexpression. RNA-seq analysis indicates that tPA in the SN is required for the upregulation of genes involved in the innate and adaptive immune responses induced by α-synuclein overexpression. Overexpression of α-synuclein in tPA Ala-KI mice, expressing only proteolytically inactive tPA, confirms that tPA-mediated neuroinflammation and neurodegeneration is independent of its proteolytic activity. Moreover, overexpression of proteolytically inactive tPA in tPA Ala-BAC mice leads to increased neuroinflammation and neurodegeneration compared to mice expressing normal levels of tPA, suggesting a tPA dose response. Finally, treatment of mice with glunomab, a neutralizing antibody that selectively blocks tPA binding to the N-methyl-D-aspartate receptor-1 (NMDAR1) without affecting NMDAR1 ion channel function, identifies the tPA interaction with NMDAR1 as necessary for tPA-mediated neuroinflammation and neurodegeneration in response to α-synuclein-mediated neurotoxicity. Thus, our data identifies a novel pathway that promotes DA-neuron degeneration and suggests a potential therapeutic intervention for PD targeting the tPA-NMDAR1 interaction.

VISTA-induced tumor suppression by a four amino acid intracellular motif.

VISTA is a key immune checkpoint receptor under investigation for cancer immunotherapy; however, its signaling mechanisms remain unclear. Here we identify a conserved four amino acid (NPGF) intracellular motif in VISTA that suppresses cell proliferation by constraining cell-intrinsic growth receptor signaling. The NPGF motif binds to the adapter protein NUMB and recruits Rab11 endosomal recycling machinery. We identify and characterize a class of triple-negative breast cancers with high VISTA expression and low proliferative index. In tumor cells with high VISTA levels, the NPGF motif sequesters NUMB at endosomes, which interferes with epidermal growth factor receptor (EGFR) trafficking and signaling to suppress tumor growth. These effects do not require canonical VISTA ligands, nor a functioning immune system. As a consequence of VISTA expression, EGFR receptor remains abnormally phosphorylated and cannot propagate ligand-induced signaling. Mutation of the VISTA NPGF domain reverts VISTA-induced growth suppression in multiple breast cancer mouse models. These results define a mechanism by which VISTA represses NUMB to control malignant epithelial cell growth and signaling. They also define distinct intracellular residues that are critical for VISTA-induced cell-intrinsic signaling that could be exploited to improve immunotherapy.

Crisdesalazine alleviates inflammation in an experimental autoimmune encephalomyelitis multiple sclerosis mouse model by regulating the immune system

Microglia/macrophages participate in the development of and recovery from experimental autoimmune encephalomyelitis (EAE), and the macrophage M1 (pro-inflammatory)/M2 (anti-inflammatory) phase transition is involved in EAE disease progression. We evaluated the efficacy of crisdesalazine (a novel microsomal prostaglandin E2 synthase-1 inhibitor) in an EAE model, including its immune-regulating potency in lipopolysaccharide-stimulated macrophages, and its neuroprotective effects in a macrophage-neuronal co-culture system. Crisdesalazine significantly alleviated clinical symptoms, inhibited inflammatory cell infiltration and demyelination in the spinal cord, and altered the phase of microglial/macrophage and regulatory T cells. Crisdesalazine promoted the M1 to M2 phase transition in macrophages (immunomodulation) and reduced neuronal necrosis (neuroprotection) in vitro. This is the first study to directly demonstrate the therapeutic effects of a microsomal prostaglandin E2 synthase-1 inhibitor in an EAE model and its ability to alter macrophage polarization, suggesting that it may be a new therapeutic option for the treatment of patients affected by multiple sclerosis and other autoimmune diseases.

Intestinal Akkermansia muciniphila complements the efficacy of PD1 therapy in MAFLD-related hepatocellular carcinoma.

Immune checkpoint inhibitors are not effective for metabolic dysfunction-associated fatty liver disease (MAFLD)-hepatocellular carcinoma (HCC) patients, and identifying the key gut microbiota that contributes to immune resistance in these patients is crucial. Analysis using 16S rRNA sequencing reveals a decrease in Akkermansia muciniphila (Akk) during MAFLD-promoted HCC development. Administration of Akk ameliorates liver steatosis and effectively attenuates the tumor growth in orthotopic MAFLD-HCC mouse models. Akk repairs the intestinal lining, with a decrease in the serum lipopolysaccharide (LPS) and bile acid metabolites, along with decrease in the populations of monocytic myeloid-derived suppressor cells (m-MDSCs) and M2 macrophages. Akk in combination with PD1 treatment exerts maximal growth-suppressive effect in multiple MAFLD-HCC mouse models with increased infiltration and activation of Tcells. Clinically, low Akk levels are correlated with PD1 resistance and poor progression-free survival. In conclusion, Akk is involved in the immune resistance of MAFLD-HCC and serves as a predictive biomarker for PD1 response in HCC.

MicroRNA-150 Deletion from Adult Myofibroblasts Augments Maladaptive Cardiac Remodeling Following Chronic Myocardial Infarction.

MicroRNA (miR: small noncoding RNA)-150 is evolutionarily conserved and is downregulated in patients with diverse forms of heart failure (HF) and in multiple mouse models of HF. Moreover, miR-150 is markedly correlated with the outcome of patients with HF. We previously reported that systemic or cardiomyocyte-derived miR-150 in mice elicited myocardial protection through the inhibition of cardiomyocyte death, without affecting neovascularization and T cell infiltration. Our mechanistic studies also showed that the protective roles of miR-150 in ischemic mouse hearts and human cardiac fibroblasts were, in part, attributed to the inhibition of fibroblast activation via the repression of multiple profibrotic genes. However, the extent to which miR-150 expression in adult myofibroblasts (MFs) modulates the response to myocardial infarction (MI) remains unknown. Here, we develop a novel 4-hydroxytamoxifen-inducible MF-specific miR-150 conditional knockout mouse model and demonstrate that the mouse line exhibits worse cardiac dysfunction after MI. Our studies further reveal that miR-150 ablation selectively in adult MFs exacerbates cardiac damage and apoptosis after chronic MI. Lastly, MF-specific miR-150 deletion in adult mice promotes the expression of proinflammatory and profibrotic genes as well as cardiac fibrosis following chronic MI. Our findings indicate a key protective role for MF-derived miR-150 in modulating post-MI responses.

A novel mouse model of upper tract urothelial carcinoma highlights the impact of dietary intervention on gut microbiota and carcinogenesis prevention despite carcinogen exposure.

Animal models of N-butyl-N-(4-hydroxy butyl) nitrosamine (BBN)-induced urothelial carcinoma (UC), particularly bladder cancer (BC), have long been established. However, the rare incidence of BBN-induced upper urinary tract UC (UTUC), which originates from the same urothelium as BC, remains elusive. The scarcity of animal models of UTUC has made it challenging to study the biology of UTUC. To address this problem, we tried to establish a novel mouse model of UTUC by treating multiple mice strains and sexes with BBN. The molecular consistency between the UTUC mouse model and human UTUC was confirmed using multi-omics analyses, including whole-exome, whole-transcriptome, and spatial transcriptome sequencing. 16S ribosomal RNA metagenome sequencing, metabolome analysis, and dietary interventions were employed to assess changes in the gut microbiome, metabolome, and carcinogenesis of UTUC. Of all treated mice, only female BALB/c mice developed UTUC over BC. Multi-omics analyses confirmed that the UTUC model reflected the molecular characteristics and heterogeneity of human UTUC with poor prognosis. Furthermore, the model exhibited increased Tnf-related inflammatory gene expression in the upper urinary tract and a low relative abundance of Parabacteroides distasonis in the gut. Dietary intervention, mainly without alanine, led to P. distasonis upregulation and successfully prevented UTUC, as well as suppressed Tnf-related inflammatory gene expression in the upper urinary tract despite the exposure to BBN. This is the first report to demonstrate a higher incidence of UTUC than BC in a non-engineered mouse model using BBN. Overall, this model could serve as a useful tool for comprehensively investigating UTUC in future studies.

Single-cell data-driven design of armed oncolytic virus and combination therapy to activate a cooperative innate-adaptive immunity against cancer.

Oncolytic viruses have been considered promising cancer immunotherapies. However, oncovirotherapy agents impart durable responses in only a subset of cancer patients. Thus, exploring the cellular and molecular mechanisms underlying the heterogeneous responses in patients can provide guidance to develop more effective oncolytic virus therapies. Single-cell RNA sequencing (scRNA-seq) analysis of tumors responsive and non-responsive to oncovirotherapy revealed signatures of the tumor immune microenvironment associated with immune response. Thus, we designed and constructed an armed oncolytic virus OV-5A that expressed five genes with non-redundant functions. OV-5A treatment exhibits robust immune response against various tumors in multiple mouse models, peripheral blood mononuclear cell (PBMC)-patient derived xenograft (PDX) model, organoid-immune cell co-culture systems and patient tissue sections by activating a cooperative innate-adaptive immune response against tumor cells. scRNA-seq analysis of complete responder and partial responder to OV-5A treatment guided the design of combination therapy of OV-5A. This data-driven approach paves a innovative way to rationalize the design of oncolytic virus and multi-agent combination therapies.

Downregulation of the NF-κB protein p65 is a shared phenotype among most anti-aging interventions.

Many aspects of inflammation increase with aging in mice and humans. Transcriptomic analysis revealed that many murine anti-aging interventions produce lower levels of pro-inflammatory proteins. Here, we explore the hypothesis that different longevity interventions diminish NF-κB levels, potentially mediating some of the anti-inflammatory benefits of lifespan-extending interventions. We found that the NF-κB protein p65 is significantly downregulated in the liver of several kinds of slow-aging mice. These included both sexes of GHRKO and Snell Dwarf mutant mice, and in females only of PAPPA KO mice. P65 is also lower in both sexes of mice treated with rapamycin, canagliflozin, meclizine, or acarbose, and in mice undergoing caloric restriction. Two drugs that extend lifespan of male mice, i.e. 17α-estradiol and astaxanthin, however, did not produce lower levels of p65. We also measured other canonical NF-κB signaling regulators, including the activators IKKα and IKKβ and the inhibitor IκB-α. We found that those regulators do not consistently change in a direction that would lead to of NF-κB inhibition. In contrast, we found that NCoR1, an HDAC3 cofactor and a transcription co-repressor that regulates p65 activity, was also downregulated in many of these mouse models. Finally, we report downregulation of three p65 target proteins that regulate the metabolic and inflammatory states of the liver (HNF4α, IL-1β, and CRP) in multiple slow-aging mouse models. Together, these data suggest that NF-κB signaling, might be inhibited in liver of multiple varieties of slow aging mice. This establishes p65 as a potential target for novel longevity interventions.

Entacapone alleviates muscle atrophy by modulating oxidative stress, proteolysis, and lipid aggregation in multiple mice models

BackgroundSkeletal muscle atrophy significantly affects quality of life and has socio-economic and health implications. This study evaluates the effects of entacapone (ENT) on skeletal muscle atrophy linked with oxidative stress and proteolysis.MethodsC2C12 cells were treated with dexamethasone (Dex) to simulate muscle atrophy. Four murine models were employed: diaphragm atrophy from mechanical ventilation, Dex-induced atrophy, lipopolysaccharide (LPS)-induced atrophy, and hyperlipidemia-induced atrophy. Each model utilized entacapone (10 mg/kg), with sample sizes: Control (9), MV (11), MV + ENT (5) for diaphragm atrophy; Control (4), Dex (4), Dex + ENT (5) for Dex model; Control (4), LPS (4), LPS + ENT (5) for LPS model; and similar for hyperlipidemia. Measurements included muscle strength, myofiber cross-sectional area (CSA), proteolysis, oxidative stress markers [uperoxide dismutase 1 (SOD1), uperoxide dismutase 2 (SOD2), 4-hydroxynonenal (4-HNE)], and lipid levels.ResultsOur findings confirm Dex-induced muscle atrophy, evidenced by increased expression of muscle atrophy-associated proteins, including Atrogin-1 and Murf-1, along with decreased diameter of C2C12 myotubes. Atrogin-1 levels rose by 660.6% (p < 0.05) in the Dex group compared to control, while entacapone reduced Atrogin-1 by 84.4% (p < 0.05). Similarly, Murf-1 levels increased by 365% (p < 0.05) in the Dex group and were decreased by 89.5% (p < 0.05) with entacapone. Dexamethasone exposure induces oxidative stress, evidenced by the upregulation of oxidative stress-related proteins Sod1, Sod2, and 4-HNE. Entacapone significantly reduced the levels of these oxidative stress markers, enhancing GSH-PX content by 385.6% (p < 0.05) compared to the Dex-treated group. Additionally, ENT effectively reduced the Dex-induced increase in MDA content by 63.98% (p < 0.05). Furthermore, entacapone effectively prevents the decline in diaphragm muscle strength and myofiber CSA in mice. It also mitigates diaphragm oxidative stress and protein hydrolysis. Additionally, entacapone exhibits the ability to attenuate lipid accumulation in the gastrocnemius muscle of hyperlipidemic mice and alleviate the reduction in muscle fiber CSA.ConclusionOur findings suggest that entacapone is a promising therapeutic candidate for muscle atrophy, functioning through the reduction of oxidative stress, proteolysis, and lipid aggregation. Future research should explore the underlying mechanisms and potential clinical applications of entacapone in muscle-wasting conditions.

Lysosome-Mitochondria Cascade Targeting Nanoparticle Drives Robust Pyroptosis for Cancer Immunotherapy.

The subcellular distribution of cargoes plays a crucial role in determining cell fate and therapeutic efficacy. However, achieving the precise delivery of therapeutics to specific intracellular targets remains a significant challenge. Here, we present a trimodular and acid/enzyme-gated nanoplatform (TAEN) that undergoes disassembly within acidic endosomes and then is cleaved by lysosomal cathepsin B to facilitate efficient and targeted transport of released cargoes into mitochondria compartments. By utilizing this nanovehicle, we successfully achieve selective sorting of photosensitizer molecules into mitochondria with a colocalization coefficient of up to 0.98, leading to the generation of reactive oxygen species stress specifically within the mitochondria for potent pyroptosis-based cancer therapy. The induction of mitochondrial stress triggers the intrinsic apoptotic pathway as well as caspase-3/gasdermin-E (GSDME) cascade, resulting in an enhanced cancer cell killing efficacy by nearly 2 orders of magnitude as compared to lysosomal stress. Furthermore, due to its superior capability to stimulate both innate and adaptive immune responses, our mitochondria-sorted nanophotosensitizer exhibits robust antitumor immune efficacy in multiple tumor-bearing mice models. This study not only provides insights into engineering nanomedicines for subcellular targeted delivery but also offers a valuable toolkit for advanced research in the field of nanobiology at subcellular resolution.

Short- and long-term pathologic responses to quartz are induced by nearly free silanols formed during crystal fracturing

BackgroundInhalation of respirable crystalline silica particles, including quartz, is associated with an increased risk of developing pathologies, including persistent lung inflammation, fibrosis, cancer, and systemic autoimmunity. We demonstrated that the nearly free silanols (NFS) generated upon quartz fracturing trigger the early molecular events determining quartz toxicity. Here, we address the involvement of NFS in driving short- and long-term pathogenic responses, including lung inflammation, fibrosis, cancer, and autoimmunity in multiple mouse models.ResultsIn vivo pulmonary responses to as-grown NFS-poor quartz (gQ) and fractured NFS-rich quartz (gQ-f) of synthetic origin were compared to two NFS-rich reference quartz dusts (Min-U-Sil 5, mQ-f). Acute and persistent inflammation, as well as fibrosis, were assessed 3 and 60 days, respectively, after administering one dose of particles (2 mg) via oropharyngeal aspiration (o.p.a.) to C57BL/6 mice. The carcinogenic potential was assessed in a co-carcinogenicity study using A/J mice, which were pre-treated with 3-methylcholanthrene (3-MC) and administered four doses of quartz particles (4 × 1 mg, o.p.a.), then sacrificed after 10 months. Autoimmunity was evaluated in autoimmune-prone 129/Sv mice 4 months after particle administration (2 × 1.25 mg, o.p.a). Mice exposed to NFS-rich quartz exhibited a strong acute lung inflammatory response, characterized by pro-inflammatory cytokine release and leukocyte accumulation, which persisted for up to 60 days. No inflammatory effect was observed in mice treated with NFS-poor gQ. Fibrosis onset (i.e., increased levels of pro-fibrotic factors, hydroxyproline, and collagen) was prominent in mice exposed to NFS-rich but not to NFS-poor quartz. Additionally, lung cancer development (tumour numbers) and autoimmune responses (elevated IgG and anti-dsDNA autoantibody levels) were only observed after exposure to NFS-rich quartz.ConclusionsCollectively, the results indicate that NFS, which occur upon fracturing of quartz particles, play a crucial role in the short- and long-term local and systemic responses to quartz. The assessment of NFS on amorphous or crystalline silica particles may help create a predictive model of silica pathogenicity.

Butyrate-producing Faecalibacterium prausnitzii suppresses natural killer/T-cell lymphoma by dampening the JAK-STAT pathway

BackgroundNatural killer/T-cell lymphoma (NKTCL) is a highly aggressive malignancy with a dismal prognosis, and gaps remain in understanding the determinants influencing disease outcomes.ObjectiveTo characterise the gut microbiota feature and identify...

Glutamine is critical for the maintenance of type 1 conventional dendritic cells in normal tissue and the tumor microenvironment

Proliferating tumor cells take up glutamine for anabolic processes, engendering glutamine deficiency in the tumor microenvironment. How this might impact immune cells is not well understood. Using multiple mouse models of soft tissue sarcomas, glutamine antagonists, as well as genetic and pharmacological inhibition of glutamine utilization, we found that the number and frequency of conventional dendritic cells (cDCs) is dependent on microenvironmental glutamine levels. cDCs comprise two distinct subsets-cDC1s and cDC2s, with the former subset playing a critical role in antigen cross-presentation and tumor immunity. While both subsets show dependence on glutamine, cDC1s are particularly sensitive. Notably, glutamine antagonism did not reduce the frequency of DC precursors but decreased the proliferation and survival of cDC1s. Further studies suggest a role of the nutrient sensing mechanistic target of rapamycin (mTOR) signaling pathway in this process. Taken together, these findings uncover glutamine dependence of cDC1s that is coopted by tumors to escape immune responses.

GENCODE 2025: reference gene annotation for human and mouse.

GENCODE produces comprehensive reference gene annotation for human and mouse. Entering its twentieth year, the project remains highly active as new technologies and methodologies allow us to catalog the genome at ever-increasing granularity. In particular, long-read transcriptome sequencing enables us to identify large numbers of missing transcripts and to substantially improve existing models, and our long non-coding RNA catalogs have undergone a dramatic expansion and reconfiguration as a result. Meanwhile, we are incorporating data from state-of-the-art proteomics and Ribo-seq experiments to fine-tune our annotation of translated sequences, while further insights into function can be gained from multi-genome alignments that grow richer as more species' genomes are sequenced. Such methodologies are combined into a fully integrated annotation workflow. However, the increasing complexity of our resources can present usability challenges, and we are resolving these with the creation of filtered genesets such as MANE Select and GENCODE Primary. The next challenge is to propagate annotations throughout multiple human and mouse genomes, as we enter the pangenome era. Our resources are freely available at our web portal www.gencodegenes.org, and via the Ensembl and UCSC genome browsers.

Abstract C041: MYC and p53 alterations cooperate through VEGF signaling to repress cytotoxic T cell and immunotherapy responses in prostate cancer

Abstract Patients with castration-resistant prostate cancer (CRPC) are generally unresponsive to tumor targeted and immunotherapies. Whether genetic alterations acquired during the evolution of CRPC impact immune and immunotherapy responses is largely unknown. Using our innovative electroporation-based mouse models, we generated distinct genetic subtypes of CRPC found in patients and uncovered unique immune microenvironments. Specifically, mouse and human prostate tumors with MYC amplification and p53 disruption had weak cytotoxic lymphocyte infiltration and an overall dismal prognosis. MYC and p53 cooperated to induce tumor intrinsic secretion of VEGF, which by signaling through VEGFR2 expressed on CD8+ T cells, could directly inhibit T cell activity. Targeting VEGF-VEGFR2 signaling in vivo led to CD8+ T cell-mediated tumor and metastasis growth suppression and significantly increased overall survival in MYC and p53 altered CPRC. VEGFR2 blockade also led to induction of PD-L1, and in combination with PD-L1 immune checkpoint blockade produced anti-tumor efficacy in multiple preclinical CRPC mouse models. Thus, our results identify a genetic mechanism of immune suppression through VEGF signaling in prostate cancer that can be targeted to reactivate immune and immunotherapy responses in an aggressive subtype of CRPC. Citation Format: Katherine Murphy. MYC and p53 alterations cooperate through VEGF signaling to repress cytotoxic T cell and immunotherapy responses in prostate cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor-body Interactions: The Roles of Micro- and Macroenvironment in Cancer; 2024 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(22_Suppl):Abstract nr C041.

Abstract 4139162: Optical coherence tomography measures mechanical contractile properties of the beating murine heart

Introduction: Optical imaging techniques can characterize properties of tissue at high temporal and spatial resolution, offering unique insights into electrically excitable tissues such as nerve and muscle. We hypothesized that the gated high-speed optical coherence tomography (OCT) could profile the contractile properties of mouse myocardial tissue and facilitate investigations of microscale mechanical function in the beating heart. Methods: C57BL/6 male mice (n=6) underwent a sternotomy while anesthetized and ventilated and were imaged at baseline with OCT. Another cohort of C57BL/6 male mice (n=6) were sacrificed and the hearts were ex vivo perfused with crystalloid using a Langendorff system. These hearts were imaged at baseline and after inhibition of contractile function with blebbistatin. OCT was performed using a custom tissue stabilizer and a Thorlabs TEL 321 OCT system, which was synchronized to physiologic signals using custom hardware and software and cardiac pacing protocols (Fig. 1A and 1B). OCT cross-sectional B-scan images (Fig. 1C) were obtained throughout multiple cardiac cycles in both in vivo and ex vivo preparations. Data were processed in MATLAB and ImageJ using the workflow in Fig. 1D. Results: Highly reproducible cross-sectional mean intensity changes were observed over the contractile cycle in vivo and ex vivo (Fig. 1E, left and middle). With mechanical contraction inhibited by blebbistatin, this cyclical intensity extinguished, linking the optical scattering change measured by OCT to myocardial contraction (Fig. 1E, right). B-scan curves averaged over multiple mice and temporally aligned with the ECG waveform (Fig. 1F) demonstrate a signal intensity waveform that reproducibly varies with the contractile cycle for both in vivo and ex vivo preparations. In vivo and ex vivo baseline waveforms both had peak intensity during systole and minimum in end-diastole (Fig. 1F). Reproducibility in ex vivo beating heart preparations without blood perfusion provides evidence that myocardial mechanical properties, not red blood cell flow, generate the intensity changes. Conclusion: Gated high-speed OCT imaging can quantify the mechanical contraction wave in the myocardium. Combined with gated OCT angiography, OCT techniques will provide high resolution imaging of both blood flow and mechanical contractility of the beating heart in small animal models of heart disease.

Fmo-4 promotes longevity and stress resistance via ER to mitochondria calcium regulation in C. elegans.

Flavin-containing monooxygenases (FMOs) are a conserved family of xenobiotic enzymes upregulated in multiple longevity interventions, including nematode and mouse models. Previous work supports that C. elegans fmo-2 promotes longevity, stress resistance, and healthspan by rewiring endogenous metabolism. However, there are five C. elegans FMOs and five mammalian FMOs, and it is not known whether promoting longevity and health benefits is a conserved role of this gene family. Here, we report that expression of C. elegans fmo-4 promotes lifespan extension and paraquat stress resistance downstream of both dietary restriction and inhibition of mTOR. We find that overexpression of fmo-4 in just the hypodermis is sufficient for these benefits, and that this expression significantly modifies the transcriptome. By analyzing changes in gene expression, we find that genes related to calcium signaling are significantly altered downstream of fmo-4 expression. Highlighting the importance of calcium homeostasis in this pathway, fmo-4 overexpressing animals are sensitive to thapsigargin, an ER stressor that inhibits calcium flux from the cytosol to the ER lumen. This calcium/fmo-4 interaction is solidified by data showing that modulating intracellular calcium with either small molecules or genetics can change expression of fmo-4 and/or interact with fmo-4 to affect lifespan and stress resistance. Further analysis supports a pathway where fmo-4 modulates calcium homeostasis downstream of activating transcription factor-6 (atf-6), whose knockdown induces and requires fmo-4 expression. Together, our data identify fmo-4 as a longevity-promoting gene whose actions interact with known longevity pathways and calcium homeostasis.

STEM-14. INTEGRATED ANALYSIS OF GLIOBLASTOMA PROGENITOR SUBTYPES REVEALS A TUMOR-DERIVED STEM CELL POPULATION WITH NEURAL AND VASCULAR POTENTIAL

Abstract Glioblastoma (GBM) exhibits a vast heterogeneity in cell types and states. Recent research highlights the reactivation of neurodevelopmental programs in GBM, contributing to this heterogeneity and, ultimately, therapy resistance. To characterize the landscape of progenitor cell types in human primary GBM, we compiled a meta-atlas of single-cell transcriptomes from seven published studies. Using a novel bioinformatic method for gene program analysis, we derived GBM gene expression meta-modules represented across multiple tumors. Our analyses validated previously described GBM gene programs and revealed novel tumor progenitor subtypes. Among these subtypes, we identified a tumor-derived stem cell population exhibiting a mixed vascular identity and transcriptional programs reminiscent of neural stem cells, which we identified as a putative neurovascular progenitor (NVP). To our knowledge, this population has not been previously described in GBM. We validated the existence of NVPs in situ using immunofluorescence from primary patient GBM samples. We then performed an enrichment for NVPs using fluorescence-activated cell sorting direct from patient tumors, followed by single-cell RNA sequencing. Examination of data from traditional model systems, including gliomaspheres and mouse GBM models, confirmed the preservation of NVP identity across systems. In vivo analysis of NVPs in multiple mouse models of GBM elucidate the influence of NVPs on GBM cell type composition, highlighting their role as multipotent progenitors. Finally, we utilized single-cell lineage barcoding to trace the progeny of patient-derived NVP cells in a novel organoid transplantation model, revealing NVP differentiation into vascular-like and neural-like cells. These data indicate that NVP cells play crucial roles in tumor plasticity and may represent a valuable therapeutic target for future exploration.

BIOM-37. EXPLORING L-TYPE AMINO ACID TRANSPORTER 1 (LAT1) AS A DIAGNOSTIC MARKER AND THERAPEUTIC TARGET IN MALIGNANT PERIPHERAL NERVE SHEATH TUMORS (MPNST)

Abstract OBJECTIVES Malignant peripheral nerve sheath tumors (MPNST) are aggressive soft tissue sarcomas that frequently arise from benign plexiform neurofibromas (PN). Approximately half of MPNST are associated with Neurofibromatosis type 1 (NF1). Despite partial elucidation of the molecular mechanisms underlying MPNST, diagnosis remains challenging, and prognosis is poor. Previously, we found that MPNST cells have elevated βIII-spectrin, a cytoskeletal protein involved in cell morphology and regulation of transporters, including L-type Amino Acid Transporter 1 (LAT1). LAT1 facilitates transport of large neutral amino acids and is upregulated in other cancer types. Our objective is to investigate LAT1 as a diagnostic marker and therapeutic target for MPNST. METHODS Gene and protein expression were determined by qPCR and WES protein analysis, respectively. The effect of shRNA knockdown of LAT1 in MPNST cells was assessed using IncuCyte proliferation assays as well as multiple mouse models. Tumor uptake of [18F] FDOPA, primarily transported by LAT1, in MPNST PDX tumors was assessed using PET/CT imaging. RESULTS LAT1 was expressed in MPNST (n=14) vs. benign PN tissues (n=6). Additionally, in human and murine MPNST cells, shRNA-mediated knockdown of LAT1 suppressed proliferation of MPNST cells in vitro and tumor growth in vivo. In a subcutaneous tumor growth model, intratumor injection of shLAT1 lentivirus reduced tumor volume to approximately 50% of control. Furthermore, uptake of [18F] FDOPA by LAT1 in MPNST PDX tumors was detected by PET/CT imaging. WU-356 PDX tissues exhibited higher LAT1 expression as well as increased uptake of [18F] FDOPA in tumors. Conversely, PET imaging showed significantly lower uptake of [18F] FDOPA in LAT1 knockdown tumors (p<0.05). CONCLUSIONS These results suggest that LAT1 promotes the growth and survival of MPNST cells. In addition, these findings demonstrate the potential use of LAT1 and the radiotracer [18F] FDOPA as a diagnostic biomarker in the management of MPNST.

In situ editing of tumour cell membranes induces aggregation and capture of PD-L1 membrane proteins for enhanced cancer immunotherapy

Immune checkpoint blockade (ICB) therapy has emerged as a new therapeutic paradigm for a variety of advanced cancers, but wide clinical application is hindered by low response rate. Here we use a peptide-based, biomimetic, self-assembly strategy to generate a nanoparticle, TPM1, for binding PD-L1 on tumour cell surface. Upon binding with PD-L1, TPM1 transforms into fibrillar networks in situ to facilitate the aggregation of both bound and unbound PD-L1, thereby resulting in the blockade of the PD-1/PD-L1 pathway. Characterizations of TPM1 manifest a prolonged retention in tumour ( > 7 days) and anti-cancer effects associated with reinvigorating CD8+ T cells in multiple mice tumour models. Our results thus hint TPM1 as a potential strategy for enhancing the ICB efficacy.