Biology subjects

Xu, Y.

Publications and source records attributed to Xu, Y..

At least 127 records · Page 7Linked to original sources

bioRxiv2026

Systematic analysis of the type VII secretion system in Streptococcus gallolyticus subsp. gallolyticus reveals genomic diversity and functional associations

Streptococcus gallolyticus subsp. gallolyticus (Sgg) is an opportunistic pathobiont associated with bacteremia, infective endocarditis, and colorectal cancer. However, the genomic diversity of this subspecies and the distribution of key virulence determinants, particularly the type VII secretion system (T7SS), remain poorly characterized. Here, we performed genomic analyses of 76 Sgg strains from diverse geographic and host origins. Core- and pan-genome analyses, multi locus sequence typing, and phylogenetic reconstruction revealed dominant sequence types (STs) that correlate with geographic origin or source of isolation. Furthermore, systematic characterization of the T7SS locus identified five new T7SS subtypes and demonstrated a strong association between T7SS subtype and ST. We further expanded the known repertoire of T7SS LXG domain-containing polymorphic toxins (LXG toxins) in Sgg substantially through genome-wide searches. Distinct distribution patterns were observed for the LXG toxins across the strains. Lastly, our data indicated that T7SS subtype was significantly associated with biofilm formation capacity of Sgg strains. Together, these findings advance our understanding of Sgg genomic diversity, reveal substantial lineage-associated variation in T7SS architecture and effector repertoires, and suggest a previously unrecognized connection between T7SS and biofilm formation in Sgg.

microbiology↗

bioRxiv2026

CellBench-LS: Benchmark Evaluation of Single-cell Foundation Models for Low-supervision Scenarios

Single-cell foundation models are trained on millions of cells, but their value when downstream labels are scarce remains uncertain. Here we compare seven such models with three established representations across clustering, batch correction, cell type annotation, gene expression reconstruction and perturbation prediction. Frozen representations are tested directly for clustering and batch correction and with lightweight task heads for few-shot prediction. CellPLM led the aggregate clustering, annotation and batch-correction comparisons, scMulan led perturbation prediction, and principal component analysis led reconstruction. Of the seven foundation models, one exceeded the strongest classical baseline in clustering, three in batch correction, four in annotation, none in reconstruction and six in perturbation prediction. Representation analyses linked reconstruction performance to preservation of linear gene relationships, but cross-model gene-relation agreement did not exceed a dimension-matched random-projection null. These results show that the utility of a cell representation depends on the information required by the downstream task.

cell biology↗

bioRxiv2026

Novel Engineered AAV Variants Demonstrate Superior Blood-Brain Barrier Penetration and Safety in Non-Human Primates

Systemic delivery of adeno-associated virus (AAV) for gene therapy of central nervous system (CNS) disorders is limited by inefficient blood-brain barrier (BBB) penetration and dose-limiting toxicity in peripheral organs, notably the liver and dorsal root ganglia (DRG)1-5. Here, we report the development of novel AAV variants via a proprietary capsid engineering platform (REACH). In non-human primates (NHPs), intravenous administration of lead variants resulted in transgene expression levels in the brain that were 600-2000 fold higher than AAV9 at the RNA level, concomitant with a 10-50 fold reduction in liver tropism and minimal off-target exposure in the heart and DRG. These engineered capsids achieve unprecedented, pan-CNS transduction with a markedly improved safety profile, representing a transformative platform for treating a broad spectrum of neurological diseases.

neuroscience↗

bioRxiv2026

Dual Control of LDL-cholesterol Levels by ANGPTL3 and ANGPTL8

BACKGROUNDInactivation of ANGPTL3 (angiopoietin-like protein 3, A3) is a proven therapeutic strategy for lowering plasma lipid levels independently of the LDL receptor (LDLR), yet the optimal approach to inactivate A3 remains unclear. A3 is proteolytically cleaved and circulates as full-length (A3-FL), N-terminal (A3-Nter) and C-terminal (A3-Cter) fragments. The specific contribution of each form of A3, and of its paralog, ANGPTL8 (A8), in modulating circulating levels of ApoB-Containing Lipoproteins (ABCLs) remain poorly defined. Clarifying these relationships will inform next-generation A3-directed therapies. METHODSWe performed liver perfusion studies to directly compare the number and composition of VLDL particles secreted from mice with and without A3. To amplify effects on cholesterol metabolism, we generated Ldlr-/- mice expressing wildtype A3 (A3-WT), A3-FL or A3-Nter, with or without co-expression of A8, and analyzed plasma lipids, circulating A3 and A8 complexes, and intravascular lipase activities. Complementary in vitro assays and structural modeling were used to assess relative endothelial lipase (EL) inhibition by A3 alone or in complex with A8. RESULTSLiver perfusion studies revealed that A3 inactivation does not alter the rates of hepatic secretion of VLDL in wildtype or Ldlr-/- mice. Inactivation of A8 alone lowered plasma LDL-cholesterol (C) levels by [~]20%, an effect dependent upon the expression of both EL and A3. Maximal inhibition of lipoprotein lipase (LPL) required co-expression of A8 plus both A3-FL and A3-Nter, indicating that A3 cleavage, in addition to A8 expression, is essential for maximal LPL inhibition. In contrast, A8 expression, but not A3 cleavage, was required for optimal EL inhibition. CONCLUSIONSA8 acts in concert with A3 to differentially modulate LPL- and EL-mediated lipolysis, which antagonizes hepatic clearance of newly-secreted atherogenic ABCLs. This mechanistic framework refines our understanding of A3-targeted lipid lowering and highlights the therapeutic potential of dual A3- plus A8-directed strategies to treat dyslipidemia and prevent atherosclerotic cardiovascular disease. Clinical perspectiveO_ST_ABSWhat is new?C_ST_ABSO_LIInactivation of A3 lowers circulating ABCL levels without altering hepatic secretion rates of VLDL-ApoB or -TG. C_LIO_LIProteolytic cleavage of A3 is required for maximal inhibition of LPL. C_LIO_LIInactivation of A8 lowers LDL-C levels through an A3- and EL-dependent, but LDLR-independent, mechanism. C_LI What are the clinical implications?O_LICombining A8 inhibition with A3-inactivating therapies offers a strategy to achieve greater reduction in LDL-C levels and atherosclerotic cardiovascular risk. C_LI

molecular biology↗

bioRxiv2026

Haplotype-resolved centromeric chromatin organization from a complete diploid human genome

Centromeres ensure proper chromosome segregation during cell division, yet the organization and regulation of centromeric chromatin within satellite DNA arrays remain incompletely understood. Here, we leverage the complete diploid human genome benchmark (T2T-HG002) to provide a detailed study of centromeric sequence and chromatin architecture on individual haplotypes. Using adaptive-sampling-enriched, ultra-long-read DiMeLo-seq, we achieve single-molecule chromatin profiling across all centromeres, revealing that along single chromatin fibers, CENP-A, the histone variant specifying centromere identity, forms multiple discrete subdomains within hypomethylated centromere dip regions (CDRs) that are flanked by H3K9me3-enriched heterochromatin. Despite underlying sequence variation, CDRs localize to sequence-homogeneous domains and maintain relatively balanced CENP-A dosage and aggregate length across all chromosomes and between haplotypes. Further, we show that bidirectional changes to centromeric and pericentromeric DNA methylation are accompanied by changes to centromeric chromatin architecture. In passaged cells with centromeric hypomethylation, subdomain boundaries are eroded, and adjacent CENP-A domains tend to merge and expand. Conversely, in pluripotent stem cells with centromeric hypermethylation, CDRs are fundamentally reorganized, such that discrete hypomethylated domains are frequently consolidated into broader contiguous tracts. These methylation-associated CDR restructuring events suggest that DNA methylation acts as a principal regulator of human centromere organization, with implications for understanding centromere plasticity, epigenetic inheritance, and chromosomal instability in development and disease.

genomics↗

bioRxiv2026

A Non-Canonical Role of SMAD4 in Regulating 3D Genome Architecture to Inhibit Lung Squamous Cell Carcinoma Development

Lung squamous cell carcinoma (LUSC) lacks clearly defined key drivers and effective targeted therapies, reflecting an incomplete understanding of its molecular pathogenesis. Here, we identify SMAD4 as a critical regulator of three-dimensional (3D) genome organization in LUSC and uncover a mechanistic link between tumor suppressor loss and oncogenic transcriptional activation. By integrating clinical datasets, genetically engineered mouse models, human and murine LUSC cell lines, and multi-omics analyses, we demonstrate that SMAD4 deficiency promotes LUSC progression by unleashing EP300-mediated enhancer-promoter looping at the SOX2 locus. Mechanistically, SMAD4 does not directly bind SOX2 regulatory elements but instead constrains chromatin looping by sequestering EP300 away from loop anchor regions. Loss of SMAD4 leads to enhanced H3K27ac deposition, aberrant SOX2 activation, and increased LUSC tumor cell proliferation. Together, these findings reveal a non-canonical role for a transcription factor (e.g., SMAD4) in regulating dysregulated 3D genome architecture to inhibit tumor development.

cancer biology↗

bioRxiv2026

Hapln1-HA signaling promotes progenitor cell proliferation and spinal cord regeneration

Adult zebrafish exhibit scarless repair and functional recovery following spinal cord injury. Their regenerative capacity is attributed to potent stem-like progenitors that mediate neuronal and glial repair. Zebrafish are thought to lack anti-regenerative extracellular matrix (ECM) components abundant in mammalian SCI, but the positive contributions of ECM to spontaneous spinal cord repair are less understood. By employing cross-species single-cell transcriptomics, we found the hyaluran modifying enzyme Hapln1 is upregulated in zebrafish progenitors but not in mouse progenitors following injury. Loss-of-function of hapln1a/b and ablation of hapln1+ cells reduce progenitor cell activation and hinder spontaneous recovery from injury. Using a series of in vivo and in vitro assays, we show that Hapln1 is required for hyaluran-cd44b mediated progenitor cell proliferation. This study reveals that, in addition to lacking anti-regenerative ECM components around SC lesions, zebrafish can also leverage pro-regenerative ECM molecules to enhance progenitor cell potency and promote repair.

neuroscience↗

bioRxiv2026

Integrated single-cell and bulk transcriptomic analysis leverages liver metastasis-related genes to develop a prognostic model for colorectal cancer patients

PurposeDifferentially expressed genes (DEGs) between colorectal cancer liver metastasis (CRLM) epithelium and primary colorectal cancer (CRC) epithelium (LMR DEGs) identified based on single-cell RNA sequencing (scRNA-seq) data may become new biomarkers for CRC prognosis. MethodsAn scRNA-seq dataset was used to describe the cellular landscape of primary CRC and CRLM and identify LMR DEGs. Prognostic LMR DEGs were identified in the bulk RNA-seq dataset. Based on the prognostic LMR DEGs, multiple machine learning algorithm combinations were compared in terms of their C-index, and the best model was selected for the construction of the LMR score. ResultsAmong the 2070 LMR DEGs, 426 prognostic LMR DEGs were ultimately obtained. The combination of the randomized survival forest (RSF) model and ridge regression had the highest C-index and was therefore used to construct a 15-gene scoring system (LMR score). In the external validation set, the 1- and 5-year AUCs of the LMR score were greater than those of the AJCC stage and other scoring systems constructed with a similar dataset. In addition, the LMR score was closely associated with factors that influence CRC outcomes, such as immune infiltration. ConclusionThe LMR score may be a reliable new biomarker for predicting the prognosis of patients with CRC.

cancer biology↗

bioRxiv2026

Polyclonal-Monoclonal Transition in Lung Squamous Cell Carcinoma Evolution

The evolutionary trajectory of lung squamous cell carcinoma (LUSC) remains poorly defined, hindering the development of effective therapies. By integrating genomic and transcriptomic sequencing from human LUSC specimens, we delineated a polyclonal-to-monoclonal evolutionary trajectory during LUSC progression. This evolutionary pattern was corroborated by single-cell RNA sequencing, which revealed consistent tumor cell heterogeneity. Specifically, the SBS5 mutational signature was enriched and correlated with poor prognosis independent of tumor stage. By further utilizing the spontaneous LUSC mouse model to identify key genomic and genetic events in LUSC progression, we observed that the JNK pathway was inhibited and that cytoskeleton-related pathways were dysregulated during LUSC development, and identified the mutations in the JNK pathway (e.g., DACT1) and cytoskeletal regulators (e.g., KIF26A). Collectively, these findings established a polyclonal-monoclonal evolution paradigm for LUSC, potentially regulated by JNK pathways, which could benefit LUSC precision therapeutics.

genomics↗

bioRxiv2026

Subunit selective modulation of GABAA receptors using pharmacogenetically tethered neurosteroids

Neurosteroids are powerful endogenous modulators of inhibition and emerging therapeutics for anxiety, epilepsy, and mood disorders, yet their actions at defined receptor subtypes and within specific neuronal populations remain poorly resolved. Here, we engineer a neurosteroid DART (Drug Acutely Restricted by Tethering) platform to deliver neuroactive steroid (NAS) activity with cellular precision and receptor-subunit selectivity. From a screen of seventeen NAS analogs, we identified seven scaffolds suitable for further engineering, and we discovered that linker attachment at the steroid C11 position uniquely preserves NAS positive allosteric modulation of GABAA receptors, whereas C2 and C17 attachment abolished activity. C11-linked NAS-DARTs slowed IPSC decay kinetics and showed variable off-target modulation of NMDA and AMPA EPSCs. The lead DART compound, YX85.1DART.2, enhanced GABA-evoked currents in neurons expressing engineered 4/{delta}-containing GABAA receptors but spared {gamma}2-containing receptors. A complementary benzodiazepine DART BZP.1DART.2 showed the opposite selectivity. Together, these tools enable cell-restricted, subunit-resolved interrogation of neurosteroid action on inhibitory microcircuits and provide a strategy to dissect how distinct GABAA receptor subclasses contribute to circuit function and therapeutic outcomes.

neuroscience↗

bioRxiv2026

Seeded aggregation of ANXA11 induces prion-like propagation, TDP-43 co-pathology and nucleocytoplasmic transport defects

Mutations in ANXA11 cause amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), yet the mechanisms linking ANXA11 dysfunction to neurodegeneration remain poorly defined. Recent cryo-EM studies revealed heteromeric ANXA11-TDP-43 filaments in patient brains, suggesting a direct pathological connection between these two ALS-associated proteins. However, whether ANXA11 possesses intrinsic amyloidogenic properties and how its aggregation relates to TDP-43 proteinopathy remain unknown. Here, we demonstrate that ANXA11 undergoes liquid-liquid phase separation and subsequently matures into amyloid fibrils through a liquid-to-solid phase transition. ANXA11 fibrils exhibit prion-like properties, including self-templating seeding activity and intercellular propagation in human iPSC-derived neurons. Strikingly, ANXA11 fibrils induces pathological conversion of TDP-43, including hyperphosphorylation, accumulation in detergent-insoluble fractions, and formation of cytoplasmic aggregates. TurboID proximity-labeling proteomics further revealed aggregation-dependent enrichment of nuclear pore complex and nucleocytoplasmic transport factors in the ANXA11 aggregate-proximal proteome. Consistently, ANXA11 aggregation was associated with nuclear envelope abnormalities, altered nucleoporin distribution, impaired mRNA export, and progressive neuronal toxicity in iPSC-derived neurons. Together, these findings establish ANXA11 as an intrinsically amyloidogenic, phase-transition-competent protein whose seeded assemblies propagate between cells, induce TDP-43 co-pathology, and are linked to nucleocytoplasmic transport defects and neuronal injury, thereby providing a mechanistic framework for ANXA11-associated ALS/FTD pathogenesis. Graphic Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=188 SRC="FIGDIR/small/713853v1_ufig1.gif" ALT="Figure 1"> View larger version (60K): org.highwire.dtl.DTLVardef@1aaad3org.highwire.dtl.DTLVardef@c51d5eorg.highwire.dtl.DTLVardef@10b2891org.highwire.dtl.DTLVardef@1945575_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIANXA11 undergoes liquid-liquid phase separation and matures into amyloid fibrils through a liquid-to-solid phase transition. C_LIO_LIANXA11 fibrils supports homotypic seeding and propagate within SH-SY5Y cells and iPSC-derived neurons. C_LIO_LIANXA11 fibrils induce TDP-43 pathological conversion, including phosphorylation and accumulation in insoluble aggregates. C_LIO_LIANXA11 aggregation is associated with nuclear pore complex remodeling, impaired mRNA export, and neuronal toxicity. C_LI In BriefANXA11 undergoes phase separation and matures into prion-like amyloid fibrils through a liquid-to-solid transition. These seeded assemblies propagate between cells and human neurons, induce TDP-43 pathological conversion, and are associated with remodeling of nuclear pore complexes, impaired mRNA export, and progressive neuronal toxicity. ANXA11 forms seeded amyloid assemblies that spread between cells, induce TDP-43 pathology, and disrupt nucleocytoplasmic transport.

neuroscience↗

bioRxiv2026

Complete genome-derived metabolic interactions reveal impact of gut ecology on human health

Metabolic interactions govern gut microbiome assembly, yet functional rules remain obscured by genomic incompleteness and fragmentation. Here, we leverage 1,150 complete genomes to construct genome-scale metabolic models, demonstrating that draft genomes introduce systemic artifacts missing critical transport functions. We observe that metabolic interactions are intrinsically driven by genomic traits and niche specialization rather than random association. We stratifies strains into four ecological groups, including active players, resource predators, utilizers, and contributors, each of which exhibits distinct metabolite exchange/competition signatures. This functional architecture is strongly coupled to secondary metabolism. Applying this framework to inflammatory bowel disease revealed distinct temporal dynamics of ecological groups across disease subtypes, where group-specific dysbiosis predicted clinical phenotypes better than whole community profiles. Furthermore, integrating metabolic and co-occurrence networks to identify keystone for classifiers as features significantly improved disease diagnosis. Our study provides novel insights into gut metabolic interaction landscape, bridging the gap between genomic potential and clinical utility for precision microbiome therapeutics.

microbiology↗

bioRxiv2026

Single-cell Landscape of T Cell Heterogeneity in Kawasaki Disease: STAT3/JAK Axis Regulates the Lineage Differentiation Bias of Th17 Cells

BackgroundKawasaki disease (KD) is a pediatric systemic vasculitis in which T-cell-mediated immune responses play a pivotal role. However, the precise dynamic evolution of T-cell subsets during disease progression remains poorly understood. MethodsSingle-cell RNA sequencing (scRNA-seq) was employed to perform high-resolution annotation of peripheral blood mononuclear cells (PBMCs) from healthy controls and KD patients, both pre- and post- IVIG treatment. T-cell developmental trajectories were reconstructed via Monocle3-based pseudotime analysis. Furthermore, the functional significance of the significant pathway was validated in a CAWS-induced KD murine model. ResultsA high-resolution single-cell landscape identified 13 distinct T-cell subtypes. Pseudotime analysis revealed a significant lineage commitment of CD4+ T cells toward a Th17 phenotype during the acute phase of KD, synchronized with the transcriptional upregulation of the STAT3/JAK signaling axis. Animal experiments further demonstrated that pharmacological inhibition of this pathway substantially attenuated inflammatory infiltration in the cardiac vasculature of KD mice. ConclusionThis study identifies the STAT3/JAK-mediated Th17 differentiation bias as a potential regulatory program associated with acute inflammation in Kawasaki disease, thereby highlighting the STAT3/JAK axis as a potential therapeutic target.

bioinformatics↗

bioRxiv2026

Paternal behavior is controlled by preoptic Trpc5 neurons

Male parenting behavior is highly conserved and activated only after offspring are born. Here we show that the behavioral switch from infanticide in virgin male mice to caregiving in wild-type sires is associated with enhanced expression and activation of Transient receptor potential channel 5 (Trpc5) in estrogen receptor (Esr1)-expressing neurons in the hypothalamic medial preoptic area (MPOA). While selective deletion of Trpc5 from Esr1MPOA neurons diminishes paternal behavior in sires, overexpression of Trpc5 in these neurons converts the otherwise infanticidal virgin males to exhibit care to pups. Mechanistically, Trpc5-dependent changes in neuronal excitability underlie fatherhood-associated Esr1MPOA neuron plasticity. Notably, Trpc5 overexpression also enhances escape behavior and elicits exploratory diving behavior, suggesting that these neurons control a broader adaptive parenting response in males. These findings establish a Trpc5-dependent MPOA neural signal as a critical regulator of paternal behavior.

neuroscience↗

bioRxiv2026

Two ancient nuclear lineages and divergent reproductive strategies drive global success of the wheat stripe rust pathogen

Understanding the mechanisms for generating and maintaining plant pathogen genetic diversity is essential for predicting disease emergence and safeguarding the food supply. Here, using population-genomic analyses of 507 global isolates of the wheat stripe rust fungus Puccinia striiformis f. sp. tritici, we uncover two nuclear lineages that diverged before modern agriculture and reinforced during wheat domestication. Distinct lineage combinations underpin three major global populations: two predominantly sympatric, homozygous lineages (nuclA-nuclA and nuclB-nuclB) and a globally dominant heterokaryotic lineage (nuclA-nuclB) of hybrid origin. This hybrid population evolves largely clonally, accompanied by recurrent somatic nuclear exchange. Identical virulence traits can arise independently via somatic nuclear exchange in clonal populations or through sexual recombination in recombining populations, revealing convergent evolutionary routes to adaptation. Together, our results provide a transformative nuclear-level perspective linking deep evolutionary history and reproductive strategy to the global success of cereal rust fungi.

genetics↗

bioRxiv2026

Tract-explainable and underexplained synchrony play complementary roles in the functional organization of the brain

Macroscale functional connectivity emerges from a complex interplay between structural wiring and non-tract-mediated mechanisms. However, the intrinsic entanglement of these contributions obscures their specific roles in brain organization. Here, we introduce a computational modeling framework to disentangle functional synchrony into two fundamentally distinct components: tract-explainable and tract-underexplained synchrony. Validated across two large-scale human cohorts (n = 1214) and an independent marmoset dataset (n = 24), this dissociation reveals an evolutionarily conserved architectural principle. We show that tract-explainable synchrony aligns closely with structural connectomes to facilitate global integration. Conversely, tract-underexplained synchrony drives local modularity and is anchored by multiscale cortical similarity, encompassing microstructural, receptor, and transcriptomic profiles. Crucially, these components gradually dissociate from sensori-motor to higher-order association cortices, with the importance of tract-underexplained synchrony progressively increasing. Furthermore, the tract-underexplained synchrony exhibits greater individual variability and demonstrates a stronger association with individual cognitive performance. Ultimately, tract-based and non-tract-mediated mechanisms serve distinct yet complementary roles, jointly shaping a functional organization that balances conserved macroscopic stability with higher-order cognitive flexibility.

neuroscience↗

bioRxiv2026

LRP6-Guided Engineering of AAV9 Variants with Enhanced Blood-Brain Barrier Penetration and Reduced Liver Tropism in Non-Human Primates

The blood-brain barrier (BBB) severely restricts the delivery of systemically administered adeno-associated virus (AAV) vectors for central nervous system (CNS) gene therapy. To overcome this limitation, we engineered a library of AAV9 capsid variants through rational design focused on the low-density lipoprotein receptor-related protein 6 (LRP6), a conserved mediator of transcytosis. A multi-tiered screening strategy, encompassing human BBB endothelial cells followed by neuronal and glial target cells in vitro, identified three lead variants (QL9-21, QL9-22, and QL9-25) with markedly enhanced transduction potential. In mice, these variants achieved a 5-28 fold increase in brain-wide gene delivery compared to AAV9, without elevating hepatic tropism. Crucially, evaluation in non-human primates (NHPs) revealed that the lead variant, QL9-21, mediated a striking 3-40 fold enhancement in viral genome delivery across all examined brain regions versus AAV9, while concurrently reducing liver accumulation by 2.6 fold. Our study establishes an LRP6-guided engineering platform that yields novel AAV9 vectors capable of efficient, species-conserved BBB penetration coupled with a favorable safety profile, representing a significant advance toward clinically translatable CNS gene therapies.

neuroscience↗

bioRxiv2026

Stromal asparagine supports tumor adaptation to oxidative phosphorylation inhibition through SLC38A4-mediated metabolic coupling

PurposePancreatic ductal adenocarcinoma (PDAC) is characterized by a nutrient-deprived and hypoxic tumor microenvironment (TME) that imposes severe metabolic stress on cancer cells. Under these conditions, tumor cells frequently activate the integrated stress response (ISR) to adapt to TME and develop resistance to therapies. However, how TME components support tumor adaptation to mitochondrial metabolic stress remains incompletely understood. Here, we aimed to identify key metabolite involved in ISR adaptation under oxidative phosphorylation (OXPHOS) inhibition and to elucidate the metabolic symbiosis between cancer-associated fibroblasts (CAFs) and PDAC cells. MethodsWe integrated transcriptomic and metabolomic analyses with functional assays. ISR activation was evaluated by assessing the phosphorylation of eIF2 (p-eIF2) following treatment with carboxyamidotriazole orotate (CTO), an Complex I inhibitor. Metabolomic profiling was used to identify metabolites involved in ISR activation alleviation. Mouse models were used to assess therapeutic responses following depletion of the identified metabolite under CTO treatment. Genetic perturbation of Slc38a4 was performed to assess its functional role in tumor cell adaptation to metabolic stress. ResultsWe identified asparagine (ASN) as a critical metabolite supplied by CAFs to PDAC cells under OXPHOS inhibition. A minimum level of ASN is required for PDAC cells to execute ISR downstream adaptation. ASN depletion significantly enhanced the anti-tumor efficacy of OXPHOS inhibition both in vitro and in vivo. SLC38A4 emerged as a potential mediator of this interaction. SLC38A4 expression was associated with c-Myc, and its loss increased the sensitivity of PDAC cells to CTO-induced metabolic stress. ConclusionOur findings reveal a CAF-tumor metabolic crosstalk in which stromal-derived ASN supports PDAC cell adaptation to mitochondrial metabolic stress. Adaptive outcome of ISR signaling depends on the availability of key metabolic substrates such as ASN. When extracellular ASN supply is limited, the ATF4-dependent adaptive program collapses, converting ISR from a pro-survival response into a therapeutic vulnerability. SLC38A4 may function as a key mediator of this metabolic coupling and represents a potential target for enhancing the efficacy of OXPHOS inhibition in PDAC.

cancer biology↗