Biology subjects

Chen, X.

Publications and source records attributed to Chen, X..

At least 253 records · Page 14Linked to original sources

bioRxiv2025

Bispecific T cell engagers control solid tumors through clonal replacement and IL2-driven effector differentiation of CD8 T-cells

Bispecific T cell engagers (TCEs) often exhibit limited efficacy in solid tumors, in part due to immunosuppressive cues in the tumor microenvironment and low expression of targetable tumor antigens. Therapeutic strategies to improve TCE target sensitivity and enhance T cell effector functions therefore have significant translational potential. Here, we engineered TCEs that induce T cell activation in vitro against the low-abundance target antigens, TRP2/Kb and DLL3. Despite in vitro activity in these models, TCE monotherapy showed limited control of tumor growth in immunocompetent mice. Leveraging this in vivo model of TCE treatment failure, we discovered that co-treatment with TCE and a CD25-biased Interleukin-2 (IL2) rescues anti-tumor activity. Further, multimodal single-cell transcriptomic and immune repertoire analyses revealed that TCE-IL2 combination therapy controlled tumors by recruiting and activating new CD8+ T cells into the tumor microenvironment. These findings demonstrate that TCE-mediated anti-tumor responses function through a CD8+ T cell clonal replacement mechanism that can be augmented by cytokine therapy. One Sentence SummaryCombining TCE therapy with IL-2 enhances TCE efficacy in aggressive small-cell lung cancer and melanoma models with low target antigen density through T cell clonal replacement and CD8+ effector T cell differentiation.

cancer biology↗

bioRxiv2025

Global Evaluation of Congenital Heart Disease-Associated Non-Coding Variants

Abstract (Summary)Genome-wide association studies (GWAS) have mapped thousands of congenital heart disease (CHD)-associated variants within non-coding regions of the genome. Non-coding variants can alter regulatory mechanisms, such as transcription factor (TF) binding control of gene expression, potentially contributing human diseases. However, with the increasing number of disease-associated variants, comprehensive functional validation remains a significant challenge. In this work, we developed a novel method called SNP Bind-n-Seq to evaluate >3,000 CHD-risk variants for allelic binding for the cardiac TFs NKX2-5, GATA4, and TBX5 in a high-throughput manner. These binding affinity data sets were coupled with a massively parallel reporter assay (MPRA) to screen CHD-risk variant genotype-dependent regulatory activity. We identified 170 variants that exhibit allelic TF binding and 187 that modulate gene expression. Combining both approaches revealed three high-confidence variants with genotype-dependent TF binding, genotype-dependent transcriptional activity, and eQTL behavior in cardiac cells. Collectively, this study provides the first combined high-throughput biochemical and functional genomic evaluation of thousands of CHD-risk variants. HighlightsO_LIAllelic binding affinity measurements of [~]9,600 variants for NKX2-5, GATA4, and TBX5 C_LIO_LIEvaluaFon of >3,000 CHD-risk variants for genotype-dependent regulatory acFvity C_LIO_LIInteracFon networks idenFfy funcFonal variants and genes involving cardiac eQTLs C_LI Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=195 SRC="FIGDIR/small/691900v2_ufig1.gif" ALT="Figure 1"> View larger version (49K): org.highwire.dtl.DTLVardef@1f2dc96org.highwire.dtl.DTLVardef@1700929org.highwire.dtl.DTLVardef@696ec4org.highwire.dtl.DTLVardef@1e724d8_HPS_FORMAT_FIGEXP M_FIG C_FIG

genomics↗

bioRxiv2025

Salt stress disrupts local auxin and COP1 gradients in Arabidopsis apical hooks

Seedling establishment is highly sensitive to environmental cues, with light serving as a principal regulator. In darkness, seedlings enter skotomorphogenesis, marked by hypocotyl elongation and apical hook formation, which helps seedlings emerge from the soil to reach the light. Here, we show that salinity impairs soil emergence of Arabidopsis thaliana seedlings by inducing a partially photomorphogenic like phenotype in darkness, characterized by reduced apical hook curvature. Within the hook, salt stress diminished differential epidermal cell elongation required to drive hook bending, and reduced both the auxin signalling maximum and PIN3 abundance on the concave side of the hook. Transcriptome analysis revealed that salt and osmotic stresses directly alter organ-specific transcriptional profiles, including the NaCl-mediated repression of B-BOX DOMAIN PROTEIN 28 (BBX28) in hooks and cotyledons. Under control conditions, BBX28 protein accumulated asymmetrically across the hook, but this pattern was lost under salinity. Notably, the ubiquitin ligase CONSTITUTIVE PHOTOMORPHOGENIC1 (COP1) exhibited an opposite gradient to BBX28, which was similarly disrupted by salt stress or by the inhibition of polar auxin transport, presenting auxin as an upstream regulator of COP1 spatial distribution. Together, these findings establish COP1 asymmetry as a novel feature of the apical hook, indicating that spatial regulation--not just absolute levels--shapes COP1 function, and reveal how salinity disrupts hormonal, transcriptional and protein networks to compromise seedling establishment under stress. Significance StatementSeedling establishment is a critical developmental transition that contributes to survival in the local environment. While light is the primary cue driving de-etiolation, how abiotic stress shapes early development in darkness remains unclear. We show that salinity induces a photomorphogenic-like response in dark-grown Arabidopsis thaliana seedlings, disrupting apical hook formation and soil emergence. This response involves disrupted asymmetrical epidermal cell elongation and auxin signalling, repression of B-BOX DOMAIN PROTEIN 28 (BBX28), and loss of a previously unrecognized spatial gradient of the key light regulator COP1 across the apical hook. Our discovery that COP1 is spatially regulated and disrupted by stress, reveals a new dimension of light signalling and developmental control, highlighting how environmental stress constrains seedling establishment.

plant biology↗

bioRxiv2025

In vitro study of the growth, reproduction and pathogenicity responses of Fusarium oxysporum f. sp. zingiberi to autotoxins from ginger

Long-term monoculture of ginger triggers Fusarium wilt, a disease caused by Fusarium oxysporum f. sp. niveum (Foz). However, the role of autotoxins in promoting pathogen growth remains unclear. Four representative autotoxins, syringic acid, coumarin, ferulic acid and 7-Hydrooxycoumarin were selected to investigate their allelopathic effects on the growth, reproduction, and virulence-associated traits of Foz. The responses of Foz to these compounds exhibited notable variations, which may be attributed to the structural differences among the autotoxins. The autotoxic compounds differentially enhanced the key pathogenic traits of Foz. Syringic acid was the most effective stimulant of mycelial growth and cell wall-degrading enzymes activity, concurrently boosting mycotoxin production via upregulating FUB3 gene expression. Ferulic acid was the most potent promoter of sporulation and biomass accumulation, whereas 7-Hydroxycoumarin most effectively stimulated conidial germination. Notably, coumarin suppressed mycelial growth but strongly induced mycotoxin synthesis in Foz. These results provide mechanistic insights into how autotoxins in ginger monoculture systems promote Fusarium wilt.

microbiology↗

bioRxiv2025

Alphaviral capsid proteins inhibit stress granule assembly via competitive RNA binding with G3BP1

Viral infection is one of the conditions that induces stress granule (SG) formation, a cellular defense mechanism that exerts antiviral effects. To counteract this host response, viruses have evolved a broad spectrum of strategies to inhibit SG formation. However, the molecular mechanisms underlying SG inhibition remain poorly understood. The nucleocapsid proteins play a critical role in virus replication and host interaction. Here, using Semliki Forest Virus (SFV) as a model, we uncover the function of the alphavirus nucleocapsid in SG inhibition. This inhibitory function depends on oligomerization mediated by an N-terminal -helix and with a positively charged intrinsically disordered region (IDR). We show that SFV capsid directly competes with G3BP1 for RNA binding, thereby disrupting G3BP1-RNA liquid-liquid phase separation (LLPS) in vitro and SG assembly in cells. This mechanism is conserved across the alphavirus family but is not shared by the nucleocapsid of SARS-CoV-2 or other endemic viruses examined. Notably, expression of a peptide from SFV capsid is sufficient to inhibit SG formation induced by Amyotrophic Lateral Sclerosis (ALS)-associated mutations, suggesting potential therapeutic applications. Our findings reveal mechanistic insight into SG modulation by the viral capsid protein and provide a possible bioengineering tool for probing SG dynamics in health and disease.

cell biology↗

bioRxiv2025

Alphaviral nonstructural protein-host RBP co-condensation as a mechanism to sustain virus replication

It has long been recognized that the intracellular replication of alphaviruses critically relies on several key host RBPs, including G3BP1/2 and FXR1/FXR2/FMR1, but how these RBPs modulate alphaviral replication and whether it would be possible to target these RBPs for antiviral treatment are less explored. Here, using SFV as a model, we report that SFV nsP3 exploits G3BP for its condensation and transforms antiviral stress granules into proviral nsP3-G3BP co-condensates. The gel-like co-condensates of nsP3 and G3BP enrich and protect viral genomic RNAs from host RNase degradation and serve as viral translational hubs to promote viral replication. The mode of nsP3-RBP co-condensation is prevalent across alphaviruses, and disruption of nsP3 condensates is an efficient antiviral approach. Thus, these findings uncover a general anti-alphavirus strategy based on the conserved reliance of nsP3-RBP co-condensation.

cell biology↗

bioRxiv2025

Development of a New Approach Method to Monitor and Modify Caffeine Metabolism Correlated to CYP1A2 Expression

New approach methodologies (NAMs) that seek to reduce reliance on animal testing require sensitive, mechanism-based assays to accurately predict human-specific metabolic responses. Caffeine, primarily metabolized by cytochrome P450 1A2 (CYP1A2), serves as an ideal probe substrate for evaluating CYP1A2 function. Here, we describe the development of an in vitro platform that combines high-sensitivity triple quadrupole multiple reaction monitoring (MRM) LC-MS analysis of paraxanthine with quantitative reverse-transcription PCR (qPCR) of CYP1A2 expression. Using human hepatocellular carcinoma-derived cell lines (HepG2 and Hep3B), we demonstrate that modulating CYP1A2 with known effectors, sulforaphane (a known CYP1A2 inhibitor), 3-methylcholanthrene (inducer), and galangin (moderate inducer), changes in both paraxanthine accumulation and CYP1A2 mRNA levels can be effectively monitored. The correlations observed between transcriptional responses and metabolic outputs validate paraxanthine as a sensitive readout of CYP1A2 function in these cell lines. Moreover, the assay remains robust across multiple experimental conditions and facilitates insights into enzyme induction or inhibition mechanisms. By providing a straightforward and scalable alternative to animal models, this approach expands the toolbox available for interrogating xenobiotic metabolism, and enzyme regulation. Ultimately, these findings highlight the utility of an integrated cell culture-based system for advancing studies of hepatic enzyme function. This platform enables investigators to readily screen and characterize compounds that influence CYP1A2-mediated metabolism, providing a straightforward, scalable, alternative to animal models.

biochemistry↗

bioRxiv2025

Ferrapoptosis: Discovery of a Hybrid Programmed Cell Death

Traditional taxonomies classify ferroptosis and apoptosis as distinct forms of regulated cell death. Here, we challenge this view by defining and validating a previously unrecognized neuronal death entity in traumatic brain injury (TBI), which we term "Ferrapoptosis". This hybrid mode of death is characterized by the coexistence of ferroptotic and apoptotic molecular and ultrastructural features within the same neuron-- ultrastructural alterations that cannot be fully characterized by any known cell death modalities. By integrating single-cell transcriptomics with multi-layered functional assays, we show that Ferrapoptosis dominates in severe injury and the acute phase, and is critically driven by mitochondrial oxidative stress. Over time, its predominance is gradually replaced by death modes in which either ferroptosis or apoptosis alone becomes the major pathway. Genome-wide CRISPR screening further identifies Smg7 as a key regulator that synchronously activates both death programs to drive the hybrid phenotype, whereas genetic or viral inhibition of Smg7 reduces Ferrapoptosis and promotes neurological recovery after TBI in mice. Our work systematically delineates a previously unrecognized form of cell death and its pathogenetic mechanisms, providing experimental evidence to refine cell death classification, and suggesting a conceptual strategy for treating complex diseases such as TBI by targeting shared regulatory nodes. HIGHLIGHTSO_LIDefined and validated a previously unrecognized cell death modality, termed "Ferrapoptosis". C_LIO_LIElucidated the ordered pattern of Ferrapoptosis across key stages of TBI pathogenesis. C_LIO_LIRevealed mitochondrial oxidative stress as the pivotal hub integrating ferroptosis and apoptosis. C_LIO_LIIdentify Smg7 as a core driver of Ferrapoptosis and a promising neuroprotective target. C_LI

cell biology↗

bioRxiv2025

Mapping galectin-3 ligands in tear fluid establishes spliceoform-dependent lacritin binding

Galectin-3 (Gal-3) is a carbohydrate-binding protein which plays crucial roles in inflammation, immune response, cell migration, autophagy, and signaling. At the ocular surface, Gal-3 is also known to crosslink transmembrane mucins across the epithelial cell glycocalyx, forming lattice structures important for barrier function. However, the biological role of Gal-3 in circulating tear fluid remains largely unexplored. Similarly, whether Gal-3 engages extracellular glycoproteins in tears to affect downstream biological processes has yet to be investigated. As increased Gal-3 levels in tears are known to correlate with ocular pathologies such as dry eye disease (DED), we sought to elucidate the Gal-3 interactome in tear fluid and uncover biological insights into the function of Gal-3 beyond adhesion to the epithelial cell surface. Here, we combined ELISA, lectin affinity enrichment, mass spectrometry (MS)-based glycoproteomics, and lectin blotting to uncover Gal-3 interactors and their associated glycoepitopes. Overall, we report nearly 100 proteins enriched from tear fluid across 3 different patients, identifying proteins involved in immune response, inflammation, and antimicrobial activity. Most notably, we report lacritin as a novel ligand for Gal-3 and demonstrate that specific glycoforms of lacritin bearing core 2 O-glycans preferentially engage with Gal-3. Lastly, we show that the Gal-3-lacritin axis is spliceoform-specific and dependent on lacritin multimerization. Taken together, this study elucidates new ligands for Gal-3 in tear film and reveals mRNA splicing and multimerization as new biochemical mechanisms that fine-tune Gal-3 binding events.

biochemistry↗

bioRxiv2025

SPARP-seq reveals poly(A) tail shortening as a hallmark of the active human translatome

The poly(A) tail at the 3 end of mRNAs plays a critical role in the synthesis of proteins, but its dynamic relationship with the translation process has been unclear. Here, using a novel single-molecule approach SPARP-seq that integrates poly(A) assessment with ribosome profiling, we demonstrate that poly(A) tail shortening is a universal hallmark of active translation in human cells. We find that most mRNAs begin translation on single ribosome (monosome) with long tails that progressively shorten as additional ribosomes are recruited. In contrast, highly expressed housekeeping genes are translated efficiently on monosomes and possess intrinsically short poly(A) tails. Furthermore, we find that translationally repressed transcripts, such as those retaining introns, are trapped on monosomes with long tails. Pharmacological inhibition of translation induces global poly(A) tail lengthening, confirming that deadenylation is coupled to ongoing translation. These findings reveal a dynamic system where the poly(A) tail integrates mRNA status with translational fate, defining the monosome as a central hub for post-transcriptional control.

molecular biology↗

bioRxiv2025

OPUS-ET: Resolving Compositional and Conformational Heterogeneities of Biomolecules in Cryo-Electron Tomography

Structural heterogeneity in biomolecules, arising from both compositional and conformational variability, limits resolution and interpretability of cryo-electron tomography (cryo-ET). Here, we present OPUS-ET, a deep learning framework that resolves multiscale heterogeneity throughout the cryo-ET workflow. OPUS-ET combines a composition decoder that captures compositional differences with a conformation decoder that models large-scale motions, thereby providing a hierarchical representation of structural heterogeneity. Starting from noisy template-matching candidates with templates of varying similarity or quality, OPUS-ET efficiently enriches target particle populations and delivers sub-nanometer in situ reconstructions in a single round. It leads to improved resolutions by up to 4.5 [A] over expert annotations or existing deep-learning approaches in four benchmark systems, and reveals continuous conformational landscapes capturing F-F flexible coupling in mitochondrial ATP synthase and tRNA-translocation intermediates in eukaryotic and bacterial ribosomes. Together, these results establish OPUS-ET as a powerful computational tool for linking particle purification, high-resolution reconstruction, and analysis of structural heterogeneity in cryo-ET, with demonstrated robustness to template quality, initial pose noise, and clustering parameters.

molecular biology↗

bioRxiv2025

An inherent T cell-activating mRNA delivery platform for in vivo CAR T generation

The clinical success of CAR T cell therapy has underscored the need for scalable, non-invasive strategies for in vivo T cell engineering. Although mRNA delivery offers a promising alternative, current lipid-based vectors lack intrinsic T cell tropism and often rely on antibody conjugation, complicating manufacturing and raising safety concerns. Here we present an inherent T cell-activating polymer-lipid nanoparticle (ERTLNPs) that enable ligand-free, efficient mRNA transfection and activation of T cells in vivo. ERTLNPs, composed of p-toluenesulfonyl arginine (RT)-functionalized polyethylenimine (denoted as PEI-RT) and helper lipids, preferentially accumulate in the spleen following systemic administration. Without exogenous stimulation, ERTLNPs intrinsically activate T cells, triggering robust mRNA expression and proliferation. Mechanistically, ERTLNPs engage the PI3K/AKT/mTOR signaling axis to reprogram T cell metabolism, promoting expansion while restraining exhaustion. Systemic delivery of mRNA encoding fibroblast activation protein chimeric antigen receptor (mFAP CAR) via ERTLNPs leads to in situ generation of functional CAR T cells, which efficiently eliminate pathological fibroblasts in models of cancer and fibrosis, with minimal off-target effects. This ligand-free, metabolically reprogramming mRNA delivery platform provides a clinically translatable approach for in vivo CAR T cell generation and broadens the landscape of non-viral immunotherapeutic engineering.

bioengineering↗

bioRxiv2025

Human assembloid model of emergent neurotropic enteroviruses

Enteroviruses (EVs) are the leading cause of viral meningitis in children. Recent outbreaks of non-polio EVs, most notably EV-A71 and EV-D68, have been associated with a polio-like paralysis known as acute flaccid myelitis (AFM). The lack of relevant models that mimic the cellular and functional responses of these human-restricted pathogens has hampered the development of effective treatments. We have previously engineered human stem cell-derived assembloids that recapitulate the neuromuscular connections underlying muscle contractions by integrating human spinal cord/hindbrain organoids (hSpO) and human skeletal muscle. Here, we used organoids and assembloids to investigate polio and non-polio EV pathogenesis. Infection of assembloids with poliovirus (PV), EV-D68 and EV-A71 resulted in loss of muscle contraction for all three viruses, which could be prevented by treatment with an antiviral agent. Yet, despite the convergence on neuronal dysfunction, the cellular targets by which each virus acted differed. More specifically, single cell transcriptomic profiling uncovered divergent cell tropisms between the EVs, and live imaging experiments revealed different modes and kinetics of cell damage. Altogether, we describe a multi-cellular model that captures viral pathogenesis in a human and circuit-relevant context.

neuroscience↗

bioRxiv2025

Copper-Free Click Chemistry Enables High-Fidelity Engineering of Mitochondria Targeted Brain-Derived Exosomes

Mitochondrial dysfunction is a hallmark of neurodegenerative and neuroinflammatory disorders, including hypertension and cardiovascular disease, yet strategies for safe and precise mitochondrial-targeted delivery remain limited. Here, we establish strain-promoted azide-alkyne cycloaddition (SPAAC) as a biocompatible, high-fidelity chemical platform for engineering mitochondria-targeted brain-derived exosomes (BR-EVs). Copper-free click conjugation of a mitochondrial-targeting ligand (e.g. Cy5-DBCO) under mild aqueous conditions preserved vesicle morphology (30-150 nm core; 120-200 nm hydrodynamic), proteomic composition, and uptake dynamics. Time-course imaging and fluorescence recovery after photobleaching (FRAP) revealed unaltered endocytic kinetics, >75 % mitochondrial colocalization, and intact organelle architecture. In vivo neuroinflammation and biodistribution analyses demonstrated immunological neutrality, strong central nervous system retention, and minimal peripheral dispersion following intracerebroventricular administration. Proteomic profiling of unlabeled Sprague-Dawley (SD) and hypertensive Dahl salt-sensitive (DSS) BR-EVs uncovered hypertension-driven enrichment of oxidative and complement pathways correlating with mitochondrial fragmentation and reactive oxygen species generation in neuronal cultures. These findings establish SPAAC-mediated ligand conjugation as a biocompatible and chemically precise approach for generating mitochondria-targeted exosomes that preserve exosomal identity, biodistribution, and signaling fidelity--advancing a foundational platform for organelle-specific delivery and mechanistic imaging in the central nervous system.

neuroscience↗

bioRxiv2025

Comparative Cilia Analysis in the Cerebral Cortices of Turtles, Mice, and Macaques

Primary cilia are centriole-derived sensory organelles found in most vertebrate cells including neurons. In the mouse neocortex, the primary cilia of pyramidal neurons are found to orient predominantly toward the pia, reflecting a reverse movement that occurs during postnatal neuronal repositioning. This study compared cilia orientation in principal excitatory neurons in the cerebral cortex across turtles, mice, and macaques to identify patterns to infer mechanisms of cortical evolution. We first developed custom MATLAB Apps to facilitate the fast identification and statistical analyses of cilia orientation. We found that generally the primary cilia of principal neurons in sparse inside-out laminated regions, including the macaque and mouse neocortex, macaque CA1, mouse entorhinal cortex and neighboring regions, and mouse piriform cortex layer III, orient toward the pia. In contrast, primary cilia in compact laminae of these species, including the macaque and mouse dentate gyrus (DG), macaque CA3, mouse piriform cortex layer II, and turtle lateral cortex manifest opposite orientations, positioning perpendicular to the laminae. These data suggest that over the course of cortical evolution, primary cilia in principal neurons evolve from initially having no preferred orientation to becoming increasingly oriented toward the pial surface. We propose a working model for cortical evolution: the placement of principal neurons progresses from minimal migration in lower vertebrates to forward migration in higher species, and ultimately to pronounced reverse soma movement in higher inside-out laminated cortices, driven by the accumulation of large neuronal populations in the outer layers after completing radial migration.

neuroscience↗

bioRxiv2025

Contrasting genomic routes to domestication in Occidental and Oriental pears

The domestication of perennial fruit trees remains poorly understood compared with annual crops, which were shaped by strong bottlenecks and elevated genetic load. Pears (Pyrus spp.) provide an ideal model for exploring how long-lived, outcrossing crops evolved under human selection. Here, we combined high-coverage whole-genome resequencing of 396 wild and cultivated accessions from Occidental and Oriental pears with analyses of nucleotide and transposable element (TE) polymorphisms to reconstruct the demographic and adaptive history of pear domestication. Demographic inferences revealed weak or no domestication bottlenecks and extensive gene flow between wild and cultivated populations. In the Occidental lineage, dessert and perry P. communis cultivars underwent independent domestications from the same wild progenitor, P. pyraster, with divergent selection linked to fruit use. In the Oriental lineage, regionally independent domestications gave rise to Chinese and Japanese P. pyrifolia cultivars, shaped by both environmental adaptation and human selection. Selection scans identified lineage- and use-specific targets related to fruit texture, metabolism, and immunity. Contrary to the classical "cost of domestication" hypothesis, cultivated pears carried fewer deleterious variants than their wild relatives, suggesting efficient purging through selection and introgression. TE insertions mirrored population structure and occasionally occurred near selected genes, indicating a limited but detectable adaptive role. Together, our findings go beyond confirming the dual origins of Pyrus domestication to reveal contrasting demographic and adaptive pathways in Occidental and Oriental pears, illustrating independent adaptive trajectories in perennial crops, where long lifespan, self-incompatibility, and recurrent introgression shape distinctive genomic outcomes.

genomics↗

bioRxiv2025

Dual Transcriptional Drivers of Immune Suppression in SACC: NOTCH1 for Cellular and MYB for Humoral Immunity

As a classic immune-"cold" tumor, salivary adenoid cystic carcinoma (SACC) represents a significant clinical challenge. Through sequencing analyses, we identify profound systemic immune dysregulation in SACC, characterized by suppressed hematopoiesis, T-cell exhaustion, compensatory myelopoiesis, and an expanded immature B-cell compartment. While lung metastases exhibit a relatively immune-engaged microenvironment enriched in tissue-resident T cells, these sites remain dominated by immature B cells. Mechanistically, this hematopoietic dysfunction originates from direct disruption of tumor-derived signaling entering the circulation, supported by detection of the SACC-signature MYB-NFIB fusion gene in peripheral plasma and blood cells. At the molecular level, we discover that IL17RB/OLIG1/NOTCH1 signaling pathways engage in multicellular crosstalk via IL33 or TP53 to suppress T-cell activation in primary tumors. Moreover, at lung metastatic sites, the MYB/MYBL2-NOTCH1-CXCL13-CXCR5 axis is blocked through CD24 or IL17RB, thereby inhibiting B-cell maturation. Single-cell transcriptomic profiling further identifies cancer stem cells (CSCs) exhibiting dual characteristics of cell-cycle activity and immune progenitor features, a finding that clarifies the fundamental mechanism by which these cells evade immune surveillance. In conclusion, we propose that stem cell camouflage constitutes the core mechanism underlying bone marrow hematopoietic dysfunction and drives immune evasion in SACC. These findings highlight the IL17RB-NOTCH1 signaling axis as a promising therapeutic target, offering potential to overcome the long-standing challenges associated with NOTCH1-targeted therapies in this malignancy. SignificanceOur novel transcriptomic analysis revealed a key G2M/CLP dual-progenitor state in SACC immunocold tumors. Tracing its origin to hematopoietic differentiation, we uncovered a NOTCH1-mediated dual-hit mechanism: restricting antigen presentation to silence T cells while partnering with MYB to block B-cell maturation. This finding pinpoints a root cause of immune evasion and establishes this pathway as a central therapeutic target. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=107 SRC="FIGDIR/small/688163v2_ufig1.gif" ALT="Figure 1"> View larger version (36K): org.highwire.dtl.DTLVardef@18c22a2org.highwire.dtl.DTLVardef@135e381org.highwire.dtl.DTLVardef@eb4eadorg.highwire.dtl.DTLVardef@7a51c4_HPS_FORMAT_FIGEXP M_FIG C_FIG Dichotomous gene mapping and single-cell interactome reveal the microenvironmental specificity of NOTCH1/MYB regulation of T/B-cell differentiation and its distinct signaling flows.

immunology↗

bioRxiv2025

Spatially Mapping the Mechanical and Structural Properties of the Seedling Uterine Fibroid-Myometrium Interface

Uterine fibroids (leiomyomas) are highly prevalent, noncancerous tumors canonically described as stiff and collagen-dense. Still, the mechanical heterogeneity within uterine fibroids and the alterations that occur at the fibroid-myometrium interface remain poorly understood. This study quantitatively maps the local mechanical, structural, and compositional properties of seedling uterine fibroids (< 1 cm) at the interface region. Relative to patient-matched myometrial tissues, uterine fibroids exhibited increased stiffness, decreased permeability, increased diffusivity, decreased hydration, greater collagen content, and distinct collagen organization. At the fibroid-myometrium interface, a band of aligned myometrial fibers immediately adjacent to the fibroid was observed, and heterogeneous spatial patterns in elastic modulus and permeability were identified. Ultimately, this study establishes foundational knowledge on the mechanics and structure of seedling uterine fibroids, facilitating future developments of clinically translatable detection tools.

bioengineering↗