Biology subjects

Tanaka, Y.

Publications and source records attributed to Tanaka, Y..

At least 19 recordsLinked to original sources

bioRxiv2026

Coffee induces chromatin structural changes associated with histone H4K16 hypoacetylation

Histone acetylation plays a critical role in the regulation of chromatin structure and gene expression. Among histone acetylation marks, acetylation of histone H4 at lysine 16 (H4K16ac) is particularly important for the maintenance of chromatin organization. We previously identified a reduction in H4K16ac following coffee treatment through mass spectrometry-based histone modification profiling. In the present study, we investigated the effects of coffee on H4K16ac levels, chromatin structure, and gene expression. Western blotting and immunofluorescence analyses confirmed that coffee treatment reduced H4K16ac levels in a cell type-dependent manner. Notably, H4K16ac was reduced in HCT116 cells, in which H3K27ac was not decreased by coffee treatment. Quantitative analysis of DAPI-stained nuclei revealed a significant increase in the coefficient of variation of nuclear fluorescence intensity following coffee treatment, indicating alterations in chromatin structure. Furthermore, integrative analysis of publicly available H4K16ac chromatin immunoprecipitation sequencing (ChIP-seq) data and previously generated RNA sequencing (RNA-seq) data revealed that H4K16ac-enriched genes exhibited transcriptional changes following coffee treatment in K562 cells, including the downregulation of MYC-associated genes. Together, these findings demonstrate that coffee treatment induces alterations in chromatin structure accompanied by H4K16 hypoacetylation and suggest a potential association between these epigenetic changes and transcriptional regulation.

molecular biology↗

bioRxiv2026

Selective Recognition, Covalent Linkage and Chemical Exchange of Artificial Cells through Engineered α-Hemolysin

Bottom-up synthetic biology aims not only to build individual artificial cells but also to connect them into higher-order systems that communicate with each other. Reported artificial cells communicate through partner recognition, physical connection, and chemical signaling that are typically implemented by separate molecular species. Here, we engineer the pore-forming protein -hemolysin (AH) with the SpyTag-SpyCatcher (ST-SC) pair to integrate partner recognition, covalent linkage between cells, and nanopore-mediated connection of their lumens into a single protein system. Fusion of ST or SC to AH preserved heptamer assembly and pore formation, and allowed the two heptamers (AH-ST and AH-SC) to be covalently joined. Cryo-electron microscopy resolved the resulting head-to-head 14-mer assembly at 2.20 [A]. Calcein leakage and single-channel recordings confirmed that the engineered pores remain functional. Mixing ST- and SC-displaying giant unilamellar vesicles (GUVs) resulted in partner-specific association between the two vesicle populations. Finally, photo-triggered release of a newly synthesized caged ethidium bromide (EtBr) in sender GUVs produced EtBr accumulation in receiver GUVs that required direct physical linkage between the two GUVs. This platform provides a modular route toward artificial-cell assemblies in which both connectivity and cargo exchange are programmable.

synthetic biology↗

bioRxiv2026

Seven gut microbiome types across two Japanese cohorts defined by co-abundance-based microbial guilds and associated with host physiology

Intensive efforts have been made to capture structural features of the gut microbiome for assessing microbiota status in relation to host health. However, gut microbiome function emerges not only from individual microbial taxa but also from their organization into ecological guilds, complicating microbiome structure-function relationships. To address this challenge, we applied a co-abundance-based analytical framework to identify gut microbiome types (GMTs) across a discovery cohort (D-cohort) and an independent validation cohort (V-cohort) of Japanese adults. Using paired fecal samples collected one month apart from 116 participants in the D-cohort, we identified 27 co-abundance groups (CAGs) based on 16S rRNA amplicon sequence variants. These CAGs defined seven GMTs (MT-1 to MT-7) with characteristic short-chain fatty acid and bile acid profiles. The GMTs showed temporal stability, with more than 70% of participants remaining in the same GMT across sampling points. The 27-CAG framework classified 404 participants in the independent V-cohort into the seven GMTs, which represented reproducible microbial ecological states across cohorts. MT-4 exhibited dysbiosis-like features, including reduced microbial diversity, depletion of Faecalibacterium and butyrate, and enrichment of inflammation-associated taxa, and was associated with higher alcohol consumption and elevated {gamma}-glutamyl transferase levels. In contrast, Bifidobacterium-enriched MT-5 was associated with favorable renal function profiles. Collectively, co-abundance-defined microbial guilds capture coherent ecological and functional states of the gut microbiota, providing a transferable framework for linking gut microbiome organization to host physiology beyond conventional taxonomy-based classification.

microbiology↗

bioRxiv2026

In-Cell Protein Crystallization via a Locally Flexible 24-mer Assembly Precursor

In-cell protein crystallization (ICPC) produces ordered protein crystals within living cells, but the mechanisms used by proteins to acquire long-range crystalline order in the cellular environment remains poorly understood. Here, we define the assembly pathway of CipB, a crystalline inclusion protein from Photorhabdus luminescens. CipB crystals formed in cells dissolve under mild acidic conditions into a predominant 24-mer species, supporting a model in which an in-cell crystal is built from a discrete 24-mer assembly precursor rather than through direct packing of smaller oligomeric states. Structural analysis of recrystallized CipB shows that the same 24-mer architecture packs into a body-centered cubic lattice, consistent with the lattice observed for the in-cell crystals. Cryo-EM and molecular dynamics analyses indicate that the 24-mer assembly precursor preserves its overall architecture while retaining local conformational flexibility at the N-terminal and surface-loop regions. Mutation analyses further link the N-terminal region to the formation of the 24-mer precursor and surface residues to lattice assembly. These observations support a stepwise crystallization model in which N-terminal flexibility facilitates the formation of an assembly-competent 24-mer precursor, whereas defined hydrophobic surface contacts subsequently organize these precursors into a long-range-ordered lattice.

biophysics↗

bioRxiv2026

Heartbeat-Related Bodily Processing Shapes Transition Patterns in Self-Related Spontaneous Thought

Spontaneous thought changes over time, yet the moment-to-moment factors shaping these changes remain poorly understood. We examined whether heartbeat-related bodily processing, operating largely outside explicit awareness, is associated with the organization of ongoing thought. Forty adults performed an auditory attention task with intermittent thought probes in which auditory events were scheduled either 200 ms after each detected R peak (synch condition) or independently of ongoing cardiac timing (asynch condition), with occasional omissions in both conditions. Heartbeat-synchronous omissions in this paradigm have been shown to induce cardiac deceleration and modulate heartbeat-evoked potentials (HEPs). The score of the heartbeat counting task (HCT) served as a behavioral index related to cardiac interoceptive accuracy. The results showed that higher HCT score was associated with a stronger synch-asynch shift toward more self-related and less task/self-unrelated thought. HEPs showed a synch-related negative shift across several thought groups, although the magnitude of this condition effect did not reliably differ among thought groups. Overall thought-group distributions were similar across conditions, while transition analyses suggested a tendency in the synch condition toward more frequent transitions linking predominantly interoceptive stimulus-dependent thought with both on-task and self-related thought. Together, these findings suggest that heartbeat-related bodily processing may influence the organization of spontaneous thought, particularly in individuals with greater cardiac interoceptive accuracy. Bodily signals may therefore act as an automatic constraint on the ongoing stream of thought, biasing which thought contents and transitions become more likely over time.

neuroscience↗

bioRxiv2026

Oncogenic NPM-ALK reprograms the TGM1 1 interactome toward oncogenic signaling and transcriptional states

Oncogenic NPM-ALK drives aberrant signaling networks that promote malignant phenotypes; however, the molecular mechanisms linking oncogenic signaling to downstream cellular programs remain incompletely understood. Among candidate regulatory factors, transglutaminase 1 (TGM1) has not been functionally characterized in this context. Here, we investigated the role of TGM1 in NPM-ALK-expressing cells by combining proximity-dependent proteomics with functional analyses. Using a TurboID- based approach, we mapped the TGM1-associated protein network and identified extensive remodeling of this network upon NPM-ALK expression. Proteomic analyses revealed that NPM-ALK reduced TGM1-associated proteins involved in genome maintenance and DNA repair, while enhancing associations with proteins linked to cytoplasmic translation and PI3K-AKT signaling pathways. Consistent with these findings, TGM1 deficiency impaired cell proliferation without significantly affecting cell viability, indicating a specific role in maximal proliferative capacity. Furthermore, proteomic and functional analyses suggested a link between TGM1 and AKT signaling pathways. Together, these findings suggest that oncogenic NPM-ALK reprograms the TGM1 interactome toward oncogenic signaling and transcriptional states, positioning TGM1 within signaling networks associated with proliferative cellular phenotypes.

molecular biology↗

bioRxiv2026

Midbody inheritance predicts re-entry into quiescence, but not lineage potential, in mouse hematopoietic stem cells

Hematopoietic stem cells (HSCs) give rise to all blood cell lineages and possess long-term self-renewal potential. HSCs undergo symmetric division for their expansion and asymmetric division to generate one HSCs and one progenitor cells which contribute to production of mature blood cells. The midbody is a structure which is formed in the center of the intercellular bridge during cytokinesis. However, the midbody is either asymmetrically inherited by one daughter cell or symmetrically released after cell division, whether these distinct patterns of midbody inheritance influence HSC fate remain poorly understood. In this study, we designed a fusion protein hmKO2 and MgcRacGAP which is a component of midbody. We then traced the midbody inheritance during cell division and the future cell fates of HSC daughters after division by time-lapse imaging. As a result, we found that the midbody release correlated with the delay of the time to the next division but not to the lineage potential of HSCs, indicating the possibility that midbody remnant plays some roles in cell cycle progression. HighlightHematopoietic stem cells exhibit a low frequency of midbody inheritance. Midbody inheritance does not affect the lineage potential of daughter cells. Midbody loss is associated with delayed entry into the next cell cycle.

cell biology↗

bioRxiv2026

Developmental Gfi1 Dynamics Define Hematopoietic Emergenceand Adult Hematopoietic Stem Cell Potency

Gfi1 regulates endothelial-to-hematopoietic transition (EHT) and hematopoietic stem cell (HSC) maintenance; however, its expression dynamics remain unclear. We generated a non-disruptive Gfi1-T2A-mScarlet reporter to track Gfi1 expression. At E10.5, reporter selectively marked EHT in the dorsal aorta, umbilical artery, and vitelline artery. At E9.5, mScarlet-positive endothelial cells were present in the umbilical and vitelline arteries but were scarce in the dorsal aorta, indicating vascular-bed-specific differences in hemogenic activation. Gfi1 was broadly expressed in fetal liver HSCs, while higher expression levels identified HSCs with enhanced multilineage reconstitution and preferential T-cell output. Although Gfi1 expression declined during fetal-to-adult maturation, a subset of adult bone marrow HSCs retained expression and exhibited superior repopulating and self-renewal capacity. Bulk and single-cell transcriptomic analyses linked this subset to dormant and fetal-associated programs, including imprinted genes. Reduced chromatin accessibility at a conserved Gfi1 +26.5kb putative enhancer correlated with developmental Gfi1 downregulation. Thus, Gfi1 dynamics define EHT onset and functionally distinct HSC stemness.

cell biology↗

bioRxiv2026

Desialylated platelets promote hepatocyte proliferation via the ERK1/2 signaling pathway

Platelets are increasingly recognized as active regulators of tissue repair and liver regeneration beyond their classical roles in hemostasis and thrombosis. Loss of terminal sialic acid from platelet surface glycoproteins, a process known as desialylation, occurs during platelet aging or activation and has been linked to platelet clearance via the asialoglycoprotein receptor (ASGPR) on hepatocytes. However, the mechanisms by which desialylated platelets (D-plts) directly stimulate hepatocyte proliferation remain poorly understood. This study aimed to elucidate the proliferative effects of D-plts on hepatocytes and to identify the underlying signaling mechanisms. D-plts were generated and co-cultured with hepatocyte models exhibiting low or absent levels of asialoglycoprotein receptor 1 (ASGPR1) expression, including HepG2 cells, HuH-7 cells, and human chemically induced liver progenitors. Hepatocyte proliferation was assessed, and the roles of platelet-derived factors and downstream signaling pathways were investigated. Co-culture with D-plts significantly increased hepatocyte proliferation in all three cell models compared with the corresponding controls. Moreover, supernatants derived from stimulated D-plts also significantly enhanced hepatocyte proliferation, suggesting that soluble platelet-derived factors contribute to this effect. Mechanistically, the proliferative effects were mediated predominantly through the ERK1/2 signaling pathway rather than the JAK-STAT pathway in both hepatocytes co-cultured with D-plts and those treated with D-plt-derived supernatants. In conclusion, our findings demonstrate that D-plts directly promote hepatocyte proliferation through an ASGPR-independent pathway, in which ERK1/2 signaling plays a central role. These results highlight a novel mechanism through which platelet desialylation may contribute to liver regeneration. Graphical Abstract(A) Desialylated platelets are readily activated and release increased amounts of EGF, promoting hepatocyte proliferation via the EGF-ERK signaling pathway. (B) Normal platelets show lower reactivity and reduced EGF release than desialylated platelets, resulting in weaker hepatocyte proliferation. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=102 SRC="FIGDIR/small/740293v1_ufig1.gif" ALT="Figure 1"> View larger version (32K): org.highwire.dtl.DTLVardef@d8eb25org.highwire.dtl.DTLVardef@135e644org.highwire.dtl.DTLVardef@1a0a924org.highwire.dtl.DTLVardef@c092e9_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗

bioRxiv2026

DNA barcoding-based paired daughter cell analysis reveals division preferences of hematopoietic stem cells

Hematopoietic stem cells(HSCs) maintain their pools by stem-stem division and produce mature blood cells through stem-progenitor or progenitor-progenitor division. A paired daughter cell(PDC) assay combined with single cell transplantation is a powerful method to compare the lineage outputs of two HSC daughter cells. However, single-cell transplantation precludes large-scale analysis of daughter-cell pairs, as only one cell can be transplanted per recipient. Here, we developed a DNA barcoding-based PDC assay to overcome this limitation, enabling simultaneous analysis of 476 daughter pairs from individual HSC divisions and revealing that daughter-cell fates are coordinated and that HSC division patterns are biased toward stem-stem and progenitor-progenitor divisions rather than stem-progenitor divisions. These findings indicate that HSC fate outcomes are directed toward symmetric division outcomes. Integration of single-cell RNA sequencing with DNA barcoding revealed a continuum of HSC states--from balanced HSCs to myeloid-biased HSCs and ultimately to a low-output HSC subset--in which progressively reduced production of mature blood cells relative to stem cell expansion, a proxy for stem-stem division bias, exhibits distinct activities of transcription factors and signaling pathways. Overall, our analysis uncovers characteristic patterns of HSC division and links stem maintenance with distinct molecular features.

cell biology↗

bioRxiv2026

Autonomic and cortical responses to heartbeat-synchronous auditory omissions during an auditory attention task

Cardiovascular regulation depends on bidirectional communication between autonomic cardiac control and cardiac afferent signals ascending to the central nervous system, forming a heart-brain loop. Cardio-auditory omission paradigms provide a non-invasive way to probe this loop in humans by withholding scheduled tones within heartbeat-contingent auditory sequences and measuring the resulting autonomic cardiac and heartbeat-related cortical responses. Previous studies have shown omission-evoked cardiac deceleration and neural responses mainly in specific contexts such as passive listening or synchrony-judgement tasks. The present study examined whether these responses also occur under the more general condition of active responses to external stimuli. We tested 40 adults in an auditory detection task in which tones were presented either 200 ms after the ECG R-peak (synch) or at pseudo-random intervals matched to each participants resting heart rate (asynch). A five-minute no-stimulation resting recording was used to generate the asynch sequence and served as the resting comparison condition. In both auditory conditions, 10% of scheduled tones were omitted, yielding synch and asynch omissions. Heartbeat-synchronous omissions induced sustained RR-interval prolongation that exceeded responses to asynch omissions, sound-present conditions, and rest. HEP amplitude was selectively enhanced in the synch-omission condition relative to all other conditions. Reaction times to the first post-omission tone were slightly delayed, whereas overall performance remained comparable across conditions. These findings show that heartbeat-synchronous auditory omissions produce a distinct autonomic-cortical response profile under active response demands, indicating that heart-brain loop dynamics continue to shape physiological responses during ongoing behaviour.

neuroscience↗

bioRxiv2026

Metastatic Osteosarcoma is Characterized by Loss of Osteoblastic Lineage Fidelity

Osteosarcoma (OS) is a rare and aggressive bone cancer with limited therapeutic progress in several decades. To characterize the transcriptional diversity of malignant cell states in OS, we analyzed single-nucleus RNA sequencing data from 24 tumors, including both primary and metastatic lesions. We identified a canonical osteoblastic cell state that predominated in primary tumor cells, but was dampened in metastases. Integrating tumor data with data from embryonic skeletal development and an experimental osteoblast differentiation model revealed that metastatic OS cells resemble poorly specified mesenchyme enriched for non-osseous programs. Clonal populations in metastatic samples acquired mesenchymal cell states distinct from paired primary tumors. Similar cell state shifts were evident in post-treatment primary tumors relative to paired pre-treatment samples. Our results suggest that localized post-treatment and metastatic OS malignant cells lose osteoblastic lineage fidelity, which may represent a unifying axis of disease evolution and reveal new therapeutic vulnerabilities.

Cancer Biology↗

bioRxiv2026

Spatiotemporal spinal integration of descending and spinal volleys in spinal motor circuits revealed by compound motor evoked potentials

Descending corticospinal and afferent pathways underlying spinally evoked motor potential both contribute to motor output, yet how their interaction at the spinal and peripheral levels is organized spatially within a muscle remains unclear. This study investigated the spatiotemporal characteristics of descending modulation of spinally evoked motor potentials by combining subthreshold transcranial magnetic stimulation (TMS) with transcutaneous spinal cord stimulation (tSCS). In Experiment 1, spinally evoked motor potentials were recorded from multiple lower-limb muscles at various interstimulus intervals (ISIs) defined relative to central conduction time (CCT). Subthreshold TMS facilitated spinally evoked motor potentials from CCT onward across all recorded muscles, with additional bilateral facilitation observed at longer ISIs. In Experiment 2, high-density surface electromyography (HDsEMG) revealed distinct intramuscular activation patterns in the tibialis anterior. The center of gravity (CoG) of TMS-induced motor evoked potentials was located more proximally than that of spinally evoked motor potentials. Notably, the CoG of facilitation maps was shifted further proximally than that of both single-stimulus responses. These findings suggest that descending and afferent inputs preferentially recruit partially distinct motoneuron pools within the same muscle. The proximal bias of facilitation indicates recruitment of additional motoneurons rather than uniform amplification of existing activity. Together, these results demonstrate that the interaction between descending and afferent inputs is both timing-dependent and spatially non-uniform, providing new insight into sensorimotor integration in the human lower limb.

neuroscience↗

bioRxiv2026

Personalized reference genome-based pipeline reveals comprehensive haplotype-resolved views of cancer genomes

Cancer genome analysis relies on standard human reference genomes, but detecting somatic alterations in highly repetitive or individual-specific regions remains challenging. We developed the Personalized Reference genome-based Cancer Genome Analysis Pipeline (PRCGAP, https://github.com/yos-sk/PRCGAP), to our knowledge, the first comprehensive pipeline integrating haplotype-resolved analyses of somatic point mutations, structural variants, copy number, and DNA methylation on personalized diploid reference genomes, with each variant phased and annotated by genomic features. We applied PRCGAP to eight tumor-normal cell line pairs and three newly collected pediatric B-cell acute lymphoblastic leukemia (B-ALL) clinical samples. PRCGAP recovered most standard reference-based variants (86.7-96.4% across variant types), validated by orthogonal short-read sequencing, while additionally detecting variants that standard references missed (23.4% of SNVs and 32.6% of SVs). Notably, PRCGAP uncovered variants in centromeric and telomeric regions, and somatic single-nucleotide variants were enriched in centromere dip regions, the putative kinetochore formation sites. Using PRCGAP outputs, we identified L1 retrotransposition source sites absent from standard references and showed that a B-ALL IGH::DUX4 fusion arose from a DUX4 pseudogene within an internal D4Z4 repeat unit, rather than the canonical full-length DUX4 gene. Within a single haplotype-resolved framework, PRCGAP opens regions of the cancer genome that are inaccessible to standard references.

bioinformatics↗

bioRxiv2026

The slow-acting G-protein Gz defines the duration of circadian rest time

Duration of nightly rest is a trait that varies between individuals, influenced by a complex interplay between multiple genetic and environmental factors. The central circadian clock that orchestrates daily rhythm in behavior/sleep resides in the suprachiasmatic nucleus (SCN). Yet, how the SCN encodes the "length" of the circadian rest phase ({rho}) remains an open question. Here we demonstrate that the unique G-protein-subtype Gz contributes to this process by sculpting the waveform of the circadian cAMP-PKA activity rhythm within the SCN. Genetic deletion and subsequent rescue of Gz expression reversibly altered the {rho} duration, shifting it from [~]10 h in Gz+/+ mice to [~]7.5 h in Gz-/-; mice, accompanied by proportional changes in peak cAMP-PKA activity duration and transcriptome remodeling in the SCN. Notably, intra-SCN cAMP activation led to behavioral rest, with Gz specifically shaping this response without affecting 24-h rhythmicity. These findings suggest that the SCN is not merely a rhythm-generator, but actively allocates the {rho}-rest period via Gz signaling.

neuroscience↗

bioRxiv2026

A Heart-on-a-Chip Microdevice with Aligned Fibers for Cardiotoxicity Assessment

Background and ObjectivesCardiovascular cells differentiated from human induced pluripotent stem cells (iPSCs), including cardiomyocytes, are valuable for evaluating human cardiac pharmacology and toxicity. Early assessment of cardiotoxicity, especially for novel drugs like anticancer agents, is essential for improving drug development efficiency and reducing costs. This study aimed to develop a highly sensitive bioassay system capable of evaluating the physiological function of human cardiac tissue in vitro. MethodsHuman iPSCs were differentiated into cardiovascular cell types (cardiomyocytes, vascular endothelial cells, and vascular mural cells) and assembled into a cardiac tissue model on aligned fiber device. This tissue was cultured dynamically to induce the formation of vascular network-like structure. By combining the fiber device with our previously developed heart-on-a-chip microdevice (HMD), we created a new model of HMD (Aligned Fiber-based HMD; AF-HMD) with improved throughput and stability. Pulsatile force changes induced by drug exposure were quantified by tracking the displacement of fluorescent microbeads within the microchannels. ResultsAF-HMD demonstrated functional responses to known cardiac agonists and toxicants, such as doxorubicin. The device also replicated clinically relevant cardiotoxic events, including the synergistic effects of trastuzumab and doxorubicin, showing marked reductions in contractile force and beat rate, mirroring clinical observations. ConclusionsThe AF-HMD system provides a sensitive and reproducible platform for evaluating cardiotoxicity in drug development. It offers a promising tool for preclinical screening, with potential applications in personalized medicine and predicting cardiotoxic risk in cancer therapy.

bioengineering↗

bioRxiv2026

Probabilistic coupling of cellular and microenvironmental heterogeneity by masked self-supervised learning

AO_SCPLOWBSTRACTC_SCPLOWSpatial omics technologies have advanced to single-cell resolution, enabling systematic analysis of tissue microenvironments alongside cellular-state heterogeneity. However, computationally defining microenvironmental states at single-cell resolution and identifying representations most informative for biological discovery remain major challenges. Here we present Mievformer, a Transformer-based masked self-supervised framework that learns microenvironmental embeddings by encoding neighboring cellular states and relative spatial configurations to parameterize the conditional distribution of continuous cell states at central spatial positions. Through InfoNCE optimization, Mievformer learns representations that capture the relative enrichment of cell states across microenvironments, formalized as a conditional density ratio, thereby enabling probabilistic inference of the coupling between microenvironmental and cellular heterogeneity. Mievformer outperformed existing methods in niche clustering on simulated spatial transcriptomics data and achieved the highest average performance across five real datasets spanning three spatial transcriptomics platforms when evaluated using DREC, a ground-truth-free metric that most strongly correlated with ground-truth performance in simulations. Beyond conventional clustering, Mievformer enables identification of cellular subpopulations based on their microenvironmental distribution and detection of gene-expression signatures associated with colocalization of specific cell populations. Together, these results establish Mievformer as a quantitatively robust and biologically informative framework for learning microenvironment representations in spatial omics.

bioinformatics↗

bioRxiv2026

A senescent iCAF-like fibroblast state governs therapy resistance in rheumatoid arthritis

ObjectivesRheumatoid arthritis (RA) synovitis frequently persists despite cytokine-targeted therapies, suggesting the existence of pathogenic stromal programs that sustain chronic inflammation independently of canonical immune pathways. Although synovial fibroblasts (SF) are increasingly implicated in treatment resistance, the pathogenic fibroblast states driving refractory disease and their therapeutic vulnerabilities remain poorly defined. MethodsWe integrated multimodal single-cell and spatial profiling of synovial tissue from 54 patients with RA with prospective treatment-response data and functional studies in human fibroblasts and experimental arthritis models. ResultsWe identified a C-X-C motif chemokine 12 (CXCL12)hi Apolipoprotein C1 (APOC1)+ fibroblast population selectively enriched in treatment-refractory synovitis. Spatial analyses demonstrated that these fibroblasts establish CXCL12-dependent plasmablast niches within inflamed synovium, resembling inflammatory cancer-associated fibroblasts (iCAF) that orchestrate immune cell recruitment in the tumor microenvironment. CXCL12hi APOC1+ fibroblasts exhibited a senescence-associated iCAF-like transcriptional program characterized by STAT3-C/EBP activation and APOC1 expression and were associated with poor response to TNF and IL-6 pathway inhibition. Mechanistically, APOC1 knockdown in RA-SF attenuated invasive mesenchymal behavior and disrupted senescence-associated inflammatory programs, identifying APOC1 as a central regulator of pathogenic fibroblast reprogramming. Importantly, genetic or pharmacological elimination of senescent cells ameliorated experimental arthritis and enhanced the efficacy of TNF blockade. ConclusionsThese findings implicate iCAF-like fibroblasts with senescent properties as a mechanistic driver of refractory RA synovitis and highlight stromal senescence programs as preclinically actionable therapeutic targets beyond cytokine inhibition.

immunology↗