bioRxiv : the preprint server for biology最新文献

{"title":"Fluorescent proteins misreport ribosome abundance under translation inhibition.","authors":"Aniket Zodage, Justin Chen, Nabigha Mogharbel, Hidde de Jong, Suckjoon Jun","doi":"10.64898/2026.09.17.752513","DOIUrl":"10.64898/2026.09.17.752513","url":null,"abstract":"<p><p>Fluorescent proteins are widely used as quantitative reporters of protein abundance in living cells. Here we show that this relationship can break down under translation inhibition: the cellular ribosomal abundance can increase while fluorescence intensity remains unchanged or even decreases. Using fluorescent reporters of ribosome abundance in <i>Escherichia coli</i> and <i>Bacillus subtilis</i>, we find that cellular fluorescence intensity quantitatively tracks ribosome abundance, inferred by RNA-to-protein ratio, under nutrient-limited growth but that is no longer true during chloramphenicol treatment. In <i>E. coli</i>, the discrepancy occurs with transcriptional and translational reporters and with both ribosomal protein and ribosomal RNA promoters. To distinguish changes in fluorescence output per reporter from changes in reporter abundance, we constructed a fluorescent protein-LacZ dual reporter in which fluorescence and an independent enzymatic estimate of reporter abundance are obtained from the same protein. Under translation inhibition, LacZ-derived reporter abundance increases whereas fluorescence intensity remains approximately constant, showing that fluorescence output per unit reporter abundance decreases. In a companion study, Bakshi and colleagues demonstrate that the effect extends to other fluorescent proteins and other translation inhibitors, indicating that it is not specific to a particular fluorescent protein or translation inhibitor. Our results show that fluorescent-protein calibration can be condition-dependent and cannot be assumed to transfer across physiological perturbations.</p>","PeriodicalId":519960,"journal":{"name":"bioRxiv : the preprint server for biology","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2026-10-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13622061/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"149122204","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"A Novel NO Donor 3A72 Activates Mitochondrial-Associated Endoplasmic Reticulum Membrane Structures to Induce Ferroptosis in Triple-Negative Breast Cancer.","authors":"Yunke Long, Yuxin Jiang, La Li, Xiufan Wu, Jiachen Weng, Ying Chen, Hongrui Liu","doi":"10.64898/2026.09.28.754991","DOIUrl":"https://doi.org/10.64898/2026.09.28.754991","url":null,"abstract":"<p><p>Triple-negative breast cancer (TNBC) remains a major clinical challenge due to a lack of effective interventions and severe drug resistance. Disrupting tumor metabolism using nitric oxide (NO) donors to drive ferroptosis represents a promising therapeutic strategy. Here, we developed 3A72, a novel furan-coumarin-based NO donor with potent anti-TNBC efficacy. <i>In vitro</i> and <i>in vivo</i> studies demonstrated that 3A72 significantly suppresses TNBC cell proliferation and restricts xenograft tumor growth. Notably, its efficacy matches the first-line drug paclitaxel but with enhanced safety and reduced systemic toxicity. Mechanistically, 3A72 induces ferroptosis in triple negative breast cancer (TNBC) cells via a synergistic dual-pathway mechanism. Initially, 3A72 releases intracellular NO to trigger cytoplasmic calcium overload, followed by high-affinity binding to both inositol 1,4,5-trisphosphate receptor (IP R) and acyl-CoA synthetase long-chain family member 4 (ACSL4). Binding to IP ₃ R provokes mitochondrial calcium overload and a massive burst of reactive oxygen species (ROS), while concurrent ACSL4 activation drives essential membrane phospholipid remodeling. Crucially, our transcriptomic and structural predictions reveal that while ACSL4 exhibits a broader distribution across organelle membranes, its functional association with IP ₃ R spatially converges at the endoplasmic reticulum membranes associated with mitochondria (MAMs). This subcellular niche closely couples IP R-mediated ROS generation with ACSL4-driven lipid remodeling, accelerating lethal malondialdehyde (MDA) accumulation and triggering irreversible ferroptosis. Overall, these findings highlight the MAMs calcium-lipid metabolic hub as a promising structural scaffold for antitumor drug development.</p>","PeriodicalId":519960,"journal":{"name":"bioRxiv : the preprint server for biology","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2026-10-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13648585/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"149170522","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"A Novel ACLY Inhibitor Induces Transcriptional and Metabolic Reprogramming to Overcome Tyrosine kinase inhibitor Resistance in Lymphoma.","authors":"Nagarajan Pattabiraman, Cosimo Lobello, David Rushmore, Shengchun Wang, Luca Mologni, Hossein Borghaei, Kavindra Nath, Wujuan Zhang, Aaron R Goldman, Reza Nejati, Mariusz Wasik, Johnvesly Basappa","doi":"10.64898/2026.09.29.755331","DOIUrl":"https://doi.org/10.64898/2026.09.29.755331","url":null,"abstract":"<p><strong>Background: </strong>Metabolic plasticity contributes to therapeutic resistance in T-cell lymphoma, but the metabolic dependencies underlying tyrosine kinase inhibitor (TKI) resistance remain poorly understood. ATP citrate lyase (ACLY), which converts citrate to cytosolic acetyl-CoA, links glucose metabolism to lipogenesis and acetylation-dependent regulation. We hypothesized that ACLY represents an actionable metabolic vulnerability in lymphoma.</p><p><strong>Methods: </strong>Structure-guided drug discovery and biochemical screening were used to identify the novel ACLY inhibitor VCB1. VCB1 was evaluated against recombinant human ACLY and in TKI-sensitive and TKI-resistant lymphoma models. Antitumor activity was assessed by proliferation, cell-cycle analysis, apoptosis, and immunoblotting, followed by integrated transcriptomic, metabolomic, and lipidomic profiling.</p><p><strong>Results: </strong>Our xenograft study showed that ACLY dependence in ALK+ALCL. We discovered novel ACLY inhibitor VCB1 directly inhibited recombinant ACLY with µM potency (IC₅₀, 0.9 µM) and strongly suppressed lymphoma cell growth. In ALK inhibitor-sensitive SUPM2, VCB1 showed an IC₅₀ of 0.299 μM and retained potency in lorlatinib-resistant SUPM2-LR1000 cells (1.62 μM), whereas BMS-303141 was substantially less active (17.46 and 22.07 μM). VCB1 similarly inhibited BTK inhibitor sensitive and resistant mantle cell lymphoma cell growth (IC₅₀, 0.28 and 0.278 μM), compared with BMS-303141 (30.5 and 37.01 μM). Multi-omics profiling revealed suppression of de novo lipogenesis, including <i>ACACA, FASN</i> , and <i>PPARGC1B</i> , together with extensive remodeling of triglyceride, phospholipid, cardiolipin, ceramide, and acylcarnitine species and increased expression of fatty-acid oxidation genes. VCB1 activated the integrated stress response (ISR), inducing <i>ATF4, PPP1R15A, DDIT3, DDIT4</i> and <i>SESN2</i> , while disrupting purine and pyrimidine metabolism and reducing nucleotide biosynthetic genes, including <i>CAD, DHODH, UCK2</i> , and <i>ATIC</i> . VCB1 further activated an ACLY-ISR-CHAC1 axis associated with reduced GCLC/GCLM expression, glutathione depletion, and a decreased GSH/GSSG ratio, indicating impaired redox homeostasis. TKI-resistant cells exhibited distinct lipid-metabolic remodeling consistent with metabolic plasticity. VCB1 also altered immune-regulatory programs involving <i>PDCD1, TIGIT, CD247, CD226, NFATC2</i> , and inflammatory cytokines. Functionally, VCB1 induced cell cycle arrest and apoptosis.</p><p><strong>Conclusions: </strong>ACLY represents an integrated metabolic and signaling vulnerability in lymphoma. VCB1 suppresses lymphoma growth across TKI-sensitive and resistant models by disrupting lipid and nucleotide metabolism, metabolic adaptation and ISR and redox homeostasis supporting ACLY inhibition as a therapeutic strategy for TKI-resistant lymphoma.</p>","PeriodicalId":519960,"journal":{"name":"bioRxiv : the preprint server for biology","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2026-10-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13648573/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"149171081","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Precision Targeting of Heparan Sulfate Proteoglycans Broadly Dysregulates Survival Signaling and Metabolic Adaptation in Pancreatic Cancer.","authors":"Nancy D Ebelt, Suvithanandhini Loganathan, Lara C Avsharian, Katarzyna Dabrowska, Nathaniel Hansen, Khyatiben V Pathak, Patrick Pirrotte, Edwin R Manuel","doi":"10.64898/2026.10.01.756059","DOIUrl":"https://doi.org/10.64898/2026.10.01.756059","url":null,"abstract":"<p><p>Treatments for pancreatic ductal adenocarcinoma (PDAC) often fail because of the cancer's ability to adapt its signaling and metabolic pathways to circumvent treatment mechanisms. PDAC hallmarks that promote treatment resistance (increased fatty acid metabolism, survival signaling, desmoplasia, and macropinocytosis) are extensively regulated by heparan sulfate proteoglycans (HSPGs) and their binding partners. The pro-tumor functions of HSPGs are controlled by site-specific cleavage of their heparan sulfate chains by mammalian heparanase; however, cleavage by bacterial heparinase III (HepIII) represses these functions and prevents downstream, pro-tumor signaling. Using tumor-targeting, attenuated <i>Salmonella typhimurium</i> (strain YS1646), we have limited HepIII transgene expression to tumor tissue. Degradation of tumor cell heparan sulfates by HepIII resulted in decreased macropinocytosis, reduced survival signaling, lipid storage depletion, and decreased expression of translational machinery. This tumor-targeting treatment has the potential to improve the efficacy of several approved pancreatic cancer therapies, which is predicted to increase patient survival.</p>","PeriodicalId":519960,"journal":{"name":"bioRxiv : the preprint server for biology","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2026-10-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13648696/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"149168761","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Acetylated PLIN2 coordinates the innate immune system and oxylipin synthesis on lipid droplets to propagate inflammation.","authors":"Charles P Najt, Mahima Devarajan, Alex Churchill, Evan Kerr, Lauren Boatner, Samuel Gerrard, Brian Fess, Candace Guerrero, LeeAnn Higgins, Stacy Gelhaus, Douglas G Mashek","doi":"10.64898/2026.09.30.755694","DOIUrl":"https://doi.org/10.64898/2026.09.30.755694","url":null,"abstract":"<p><p>Research has implicated lipid droplets (LDs) and their associated proteins in inflammation and the innate immune response. The key LD protein Perilipin 2 (PLIN2) is tightly correlated with lipid storage and inflammation, but little is known about the mechanisms underlying its proinflammatory effects. Here, we show that PLIN2 is required for the inflammatory response to mitogens and the recruitment of pro-inflammatory proteins to the surface of LDs. PLIN2 directly binds arachidonic acid and interacts with key oxylipin synthetic enzymes such as PLA2 and COX2 to promote LD-localized oxylipin synthesis and propagate inflammatory signaling. Moreover, LPS induces PLIN2 hyperacetylation, and acetylation-dead PLIN2 mutants lose their proinflammatory properties in the presence of an inflammatory stimulus, as if PLIN2 were ablated. Thus, acetylated PLIN2 forms a key metabolon on LDs that is essential for the response and amplification of proinflammatory signaling.</p>","PeriodicalId":519960,"journal":{"name":"bioRxiv : the preprint server for biology","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2026-10-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13648695/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"149169302","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"A Novel Biofilm Amyloid Ligand for Caspase-11 Drives Hyperactivation in Dendritic Cells.","authors":"Shrutika Mintri, Guangnan Hu, Anukriti Mathur, Leah M Whiteman, Alexandre Dinis, Brian Le, Kaitlyn Grando, Patience Shumba, Vijay A Rathinam, Egil Lien, Çağla Tükel, Katherine A Fitzgerald, Stefania Gallucci","doi":"10.64898/2026.09.29.755159","DOIUrl":"10.64898/2026.09.29.755159","url":null,"abstract":"<p><p>Bacterial biofilms drive persistent infection and chronic inflammation, yet host sensing of biofilm-derived ligands remains poorly understood. Here we identify curli amyloid, an integral component of <i>Enterobacteriaceae</i> biofilms, as a novel activator of the non-canonical inflammasome in human and mouse dendritic cells (DCs). Curli both primes and activates the inflammasome, directly binding caspase-11 to engage NLRP3 and drive robust IL-1β maturation without pyroptosis, establishing curli as a previously unrecognized trigger of DC hyperactivation. Mechanistically, this non-lytic signaling is marked by limited gasdermin D oligomerization without mitochondrial localization and NINJ1-dependent membrane rupture. Functionally, curli-hyperactivated DCs show enhanced migration to the draining lymph nodes <i>in vivo</i>, suggesting consequent augmented adaptive immunity. Finally, curli-containing biofilms also elicit IL-1β secretion without cell death, identifying bacterial amyloid as a central driver of non-lytic inflammasome activation during immune recognition of biofilms.</p>","PeriodicalId":519960,"journal":{"name":"bioRxiv : the preprint server for biology","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2026-10-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13648619/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"149171244","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"A 4D GelMA-Cad/Gelatin Hydrogel for High-Resolution DMD Bioprinting of Vascularized Soft Tissue Models.","authors":"Jason X Wang, Claire A McClain, Evan Krystofiak, Holly E David, Nicole T Marguerite, W Ryland Hoskins, Haley F Sutphen, Jonathan A Kropski, A Scott McCall, Brian O'Grady","doi":"10.64898/2026.10.01.756011","DOIUrl":"10.64898/2026.10.01.756011","url":null,"abstract":"<p><p>Digital micromirror device (DMD)-based bioprinting enables high-resolution fabrication of hydrogel-based tissue constructs, but print fidelity and hydrogel mechanics are typically coupled through polymer concentration, light exposure, and crosslinking density. This creates a materials constraint for vascular bioprinting, where constructs must preserve microscale architecture during fabrication while supporting multicellular remodeling, lumen formation, and long-term tissue organization. To meet these requirements, we developed a 4D GelMA-Cad/gelatin hydrogel with time-dependent mechanics that maintains print fidelity while providing a soft, cell-permissive matrix. GelMA-Cad provides a covalently photocrosslinked, bioactive network containing an N-cadherin-mimetic peptide, while uncrosslinked gelatin serves as a transient matrix-modifying component that diffuses from the hydrogel after fabrication, producing further softening without bulk degradation of the GelMA-Cad network. We characterized hydrogel chemistry with NMR, quantified time-dependent changes in stiffness, visualized pore morphology with cryo-SEM, and evaluated the platform in retinal and pulmonary constructs. The 4D hydrogel supports 3D-patterned accuracy, cell viability, inner blood-retinal barrier vasculature, and vascularized lung-alveolar tissue models. Single-cell RNA-sequencing of the pulmonary vascular-alveolar model demonstrated compartment-specific transcriptional responses, including enrichment of lung capillary-associated endothelial features and AT1-like epithelial states. Overall, this platform decouples DMD print fidelity from post-print matrix mechanics for vascularized soft tissue bioprinting and tissue modeling.</p>","PeriodicalId":519960,"journal":{"name":"bioRxiv : the preprint server for biology","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2026-10-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13648744/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"149166965","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Red states modulate energy localization between the antenna and trap in PSI.","authors":"Marvin Asido, Halima Khatun, Anton Khmelnitskiy, Mike Reppert, Gabriela S Schlau-Cohen, Yuval Mazor","doi":"10.64898/2026.10.01.756128","DOIUrl":"10.64898/2026.10.01.756128","url":null,"abstract":"<p><p>The core antenna of the photosystem I (PSI) complex of oxygenic photosynthesis operates at near perfect quantum efficiency, making it one of the most efficient energy conversation devices in nature. The presence of low energy or red chlorophylls (chls) is nearly universal in this core antenna, and these red chls are known to extend the light harvesting capacity and control the rate of energy migration in PSI. Surprisingly, the significance of the structural position of the red chls in PSI remains unknown. In this study, we introduced mutations that generate specific red sites in the PSI core antenna, consistent with those observed in natural species. Using CryoEM, we characterized the structural changes at these red sites. Using transient absorption spectroscopy, we measured the effects of red chlorophyll at specific sites on energy transfer and trapping in PSI to identify their functional significance. While one site funneled energy to the reaction center, the other slowed equilibration in the antenna. These opposing effects indicate different functions are associated with the different red chlorophyll location, reconciling previous seemingly incompatible suggestions as to their functional role.</p>","PeriodicalId":519960,"journal":{"name":"bioRxiv : the preprint server for biology","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2026-10-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13648685/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"149169305","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"The engagement of layer 6 corticothalamic neurons in somatosensation.","authors":"Elaida D Dimwamwa, Nelson H Chang, Christian Waiblinger, Garrett B Stanley","doi":"10.64898/2026.08.11.744301","DOIUrl":"https://doi.org/10.64898/2026.08.11.744301","url":null,"abstract":"<p><p>The corticothalamic neurons from layer 6 (L6CT) of primary sensory cortices provide extensive input to the thalamus in addition to projecting across layers intracortically, positioning them to play a key hypothesized role in shaping thalamocortical signaling. With the expansion of tools for precise functional identification of L6CT neurons during in-vivo electrophysiology, increasing evidence highlights L6CT neurons as dynamic gain modulators of thalamocortical sensory responses. However, much of the work to date has been conducted under anesthesia and not in the context of awake and/or behaving animals. In this study, we show in the awake mouse that L6CT neurons convey information about the ascending sensory input, the timing of which is fast enough to contribute to the sensory response of neurons throughout the thalamocortical circuit. Overall, L6CT neurons robustly encode the presence vs absence of a sensory stimulus but are relatively weak encoders of the fine details. Benchmarked against the activity of other excitatory cortical neurons, we also provide evidence for L6CT neurons as predictors of behavioral outcome during a trained detection task. Taken together, the results in this study potentially tie L6CT neurons to behavior in tactile detection, one of the most fundamental functional roles of the pathway.</p><p><strong>Significance statement: </strong>Layer 6 corticothalamic (L6CT) neurons provide one of the major feedback pathways from the sensory cortex to the thalamus, yet the extent to which their role in sensory processing remains poorly understood. We show that L6CT neurons in mouse somatosensory cortex robustly signal the presence of tactile stimuli but are comparatively weak encoders of stimulus strength. During a sensory detection task, their responses strongly reflect whether the animal detects the stimulus and predict behavioral outcome better than other cortical excitatory neurons. These findings suggest that L6CT neurons are specialized less for representing fine sensory detail and more for signaling the occurrence and behavioral relevance of sensory events, providing insight into when and how corticothalamic feedback participates in perception.</p>","PeriodicalId":519960,"journal":{"name":"bioRxiv : the preprint server for biology","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2026-10-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13648358/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"149166104","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"High-Throughput Morphological Profiling of Patient-Derived Human Liver Organoids for Hepatotoxicity Hazard Identification and Compound Prioritization.","authors":"Sophia R Meyer, Max A Garcia, Neil Zhao, Samantha F Boisvert, Matthew McConnachie, Charles J Zhang, Benjamin S Halligan, Andrew M Tidball, Andrew Tucker, Josepha Kaganove, Jesse W Wotring, Joel A Yates, Diana Flores, Robert J Fontana, Jonathan Z Sexton","doi":"10.64898/2026.09.29.755355","DOIUrl":"10.64898/2026.09.29.755355","url":null,"abstract":"<p><p>Drug-induced liver injury (DILI) remains a major challenge in drug development because existing in vitro models poorly predict clinical hepatotoxicity. We coupled patient-derived human liver organoids (HLOs) with high-content imaging and automated machine learning (ML) to detect hepatotoxicity-associated phenotypes by morphological profiling. HLOs from six DILI patients and one healthy donor (n = 7) retained donor-specific baseline phenotypes in hepatocyte maturity, metabolism, and morphology. ML models trained per line on MAD-robustized, well-level-aggregated median features achieved compound-level AUROCs of 0.88 (95% CI 0.86-0.90) to 0.91 (95% CI 0.89-0.93) and balanced accuracies of 0.81-0.84 in distinguishing 54 hepatotoxicants from 20 non-hepatotoxic controls. Averaging predictions across all seven lines at an optimized threshold gave 94.4% sensitivity (95% CI 84.6-98.8%) and 85.0% specificity (95% CI 62.1-96.8%), substantially outperforming HLO cytotoxicity assays and cancer cell line profiling. Lines were moderately-to-highly concordant (Pearson r = 0.66-0.85), and benchmark concentration modeling revealed reproducible inter-line differences in phenotypic sensitivity (median 4.0-fold, several compounds exceeding 20-fold). Exposure anchoring of benchmark concentrations to human-equivalent plasma concentrations, using in vitro mass-balance dosimetry and curated plasma free fractions, placed organoid points of departure within 100-fold of clinical C_max for 60 of 66 compounds (23-fold residual offset, 95% CI 12-45), and placed two of the three misclassified negative controls an order of magnitude above clinical C_max. Sulindac injury signatures were significantly enhanced by TNFα co-treatment, demonstrating capacity to detect pro-inflammatory-mediated DILI. This platform preserves inter-line variability while enabling reproducible, scalable multi-line consensus hazard classification; because compounds were tested over a common nominal concentration range, the readout defines hepatocellular hazard, which must be combined with exposure as an independent input to estimate clinical DILI risk.</p>","PeriodicalId":519960,"journal":{"name":"bioRxiv : the preprint server for biology","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2026-10-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13648574/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"149168422","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}