Biology subjects

Cao, Y.

Publications and source records attributed to Cao, Y..

At least 109 records · Page 6Linked to original sources

bioRxiv2025

Convergent utilization of APN receptor by hedgehog merbecoviruses

Dipeptidyl peptidase-4 (DPP4) and angiotensin-converting enzyme 2 (ACE2) are well-established receptors for merbecoviruses, yet the receptor usage of merbecoviruses of European and Asian hedgehogs (EriCoVs) remains unknown. Here, by testing hedgehog orthologs of known coronavirus receptors, we identify hedgehog aminopeptidase N (APN) as a functional receptor for EriCoVs. Analysis of 94 APN orthologs indicates that EriCoVs have a limited host range, primarily utilizing hedgehog APN and, to a lesser extent, APN from certain felids, shaped by specific determinants at the virus-receptor interface. Cryo-EM reveals an APN-binding mode distinct from those used by alpha- and deltacoronaviruses. Functional assays indicate that hedgehog transmembrane serine protease 2 (TMPRSS2) enhances spike activation and promotes pseudovirus entry. Neutralizing antibodies targeting RBD and APN were developed and could effectively block EriCoV pseudovirus entry and propagation. These findings reveal an unexpected convergent evolution of APN utilization among merbecovirus, establishing a foundation for risk assessment and countermeasure development. Graphic abstractConvergent utilization of hedgehog APN by EriCoVs O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=115 SRC="FIGDIR/small/676546v1_ufig1.gif" ALT="Figure 1"> View larger version (48K): org.highwire.dtl.DTLVardef@1ea334aorg.highwire.dtl.DTLVardef@cd53bforg.highwire.dtl.DTLVardef@352102org.highwire.dtl.DTLVardef@a1eed_HPS_FORMAT_FIGEXP M_FIG C_FIG

molecular biology↗

bioRxiv2025

Human Cytomegalovirus Infection of Primary Human Oral Keratinocytes Induces Intermediate Keratinocyte Differentiation and an Altered Innate Immune Response

The oral mucosa is central for human cytomegalovirus (HCMV) acquisition. However, the initial infection events in the oral cavity, including how HCMV modulates oral cells to establish infection and spread, are supported by limited experimental data and therefore remain poorly understood. We investigated HCMV infection in cultures of primary human oral keratinocytes (HOKs) as a model to study HCMV infection in the oral mucosa using the TB40E-Gfp and MOLD strains of HCMV. Both viral strains successfully infected and replicated in HOKs as demonstrated by the appearance of cytopathic effects, full expression of viral transcripts, and the shedding of infectious virus. To investigate how HCMV modulates the transcriptome of primary HOKs, we performed single cell RNA sequencing (scRNA-seq) which revealed 11 subtypes of cells. Furthermore, infection resulted in significant changes in host gene expression. HCMV upregulated genes involved in immune responses, cell cycle regulation, cancer-related pathways, neuronal and synaptic functions, metabolism, stress responses, molecular chaperones, and vesicular trafficking which might be critical for viral protein expression and assembly. HCMV also increased the expression of genes related to the extracellular matrix, cell adhesion, microtubules, signal transduction, kinases, and transcriptional regulation. Conversely, HCMV downregulated genes associated with inflammation and immune responses, cell cycle control and apoptosis, cell adhesion and migration, as well as signaling pathways, growth factors, ion channels, transporters, and transcription factors when compared to uninfected cells. These findings suggest that HCMV modulates a wide range of host cellular pathways to create a favorable environment for its replication. HCMV also induced changes in HOK differentiation genes downregulating basal state genes and upregulating intermediate genes and select terminal differentiation genes, indicating HCMV might be driving HOK differentiation towards a dysregulated intermediate phenotype while inhibiting their terminal differentiation to maintain a cell state capable of sustaining viral replication. Using SciViewer, our recently developed scRNA-seq data visualization tool, we determined that as the infection progressed from low to high viral transcript accumulation, HCMV infection upregulated E2F targeted genes, some antiviral and immune responses as well as autophagy and cellular stress responses, while downregulating interferon-stimulated genes, immune and antiviral response genes, pyroptosis, inflammatory cell death, and membrane remodeling and viral entry genes. Overall, our study shows that primary human HOKs can be used as a model to study HCMV infection in the oral mucosa and that HCMV drives dysregulated intermediate oral keratinocyte differentiation and altered immune responses which likely facilitate viral replication in the oral mucosa and spread into the rest of the host.

molecular biology↗

bioRxiv2025

spa-ChIP-seq enables the comprehensive profiling of chromatin

Chromatin immunoprecipitation followed by sequencing (ChIP-seq) is widely used to study the genomic localization of DNA-associated proteins. However, conventional protocols include multiple manual steps that can introduce inconsistency and limit scalability, thereby restricting the inclusion of appropriate replicates and controls. Although the introduction of liquid handling platforms has improved reproducibility, most existing efforts have automated only a subset of the workflow, and extending automation to efficiently map non-histone proteins, such as chromatin regulators, remains challenging. Here, we present a fully automated implementation of our previously developed single-pot ChIP-seq protocol (Texari et al. 2021), named spa-ChIP-seq, which enables scalable processing of 8 to 96 ChIP-seq samples from crosslinked cells to sequencing-ready library in approximately three days with an estimated cost of $70 per sample. Benchmarking spa-ChIP-seq against manual ChIP-seq performed in parallel demonstrates comparable signal-to-noise ratio between the two workflows. Using spa-ChIP-seq, we systematically evaluate multiple parameters including shearing and crosslinking conditions, buffer compositions, and the ratio of antibody to cell-number. We find, for the first time to our knowledge, that weaker genomic localization signals are sensitive to changing the antibody to cell-number ratio, whereas the stronger signals remain unaffected. This finding underscores the importance of maintaining consistent antibody-to-cell-number ratio for comparative studies, such as treatment responses or chromatin-QTL mapping. The spa-ChIP-seq protocol is publicly available, including deck setups, operational parameters, and scripts. We envision that this robust, cost-efficient protocol will facilitate high-throughput, reproducible ChIP-seq analyses, supporting large-scale studies of antibody validation, compound screening, population genomics, and diagnostic frameworks.

genomics↗

bioRxiv2025

Improved identification of peptides, cross-link sites, and modification sites by Target-enhanced Accurate Inclusion Mass Screening (TAlMS)

Chemical cross-linking of proteins coupled with mass spectrometry provides structural insights by identifying cross-linked peptide pairs, abbreviated as cross-links. Presently, cross-link identification suffers from ambiguity and poor sensitivity, because they are typically of lower abundance and consequently of lower MS2 quality than linear peptides present in the same sample. Here, we present Target-enhanced Accurate Inclusion Mass Screening (TAIMS), a meticulously optimized targeted mass spectrometry method. TAIMS significantly improved the quality of MS2 as indicated by fragment ion coverage (FIC) and other metrics. From data-dependent acquisition (DDA) to TAIMS, high-FIC cross-links increased by 401 or 678 on yeast ribosome or E. coli lysate, respectively, or from about 40% to around 90%. As a result, TAIMS substantially enhanced the accuracy of cross-link site localization and mitigated sensitivity loss in cross-link identification from large database searches. Enhanced identification sensitivity of TAIMS is further evidenced by its capacity to recover genuine cross-link identifications from data that would typically be discarded. On cross-linked E. coli lysate, 10.5% (284/2711) of the inclusion-list entries generated from unidentified cross-link-spectrum matches gained identity through TAIMS. Of these, 230 were linear peptides and 54 were cross-links, including 10 inter-molecular cross-links missed entirely by DDA. Additionally, we demonstrate that TAIMS is a general method for identification of low-abundance, post-translationally modified peptides. On a mouse brain sample, TAIMS increased the number of phosphopeptides identified with accurate phosphosite assignment by 67%. These findings indicate that TAIMS holds broad applicability in proteomics.

biochemistry↗

bioRxiv2025

Visualising sub-second dynamics of nanoparticle extravasation in vivo

Altered vascular integrity is a hallmark for many diseases. Monitoring vascular permeability, including its extent and underlying transport pathways, is thus important for advancing our understanding of disease mechanisms towards innovative diagnosis and treatment. Here we show real-time fluorescence imaging using lanthanide-based upconversion nanoparticles as contrast agents to visualise subtle changes to vascular permeability in vivo. Based on simultaneous confocal imaging of the vasculature alongside single particle tracking in the wide-field at video rate, we performed high-throughput surveillance across live zebrafish larvae to pinpoint locations of potential nanoparticle extravasation, achieving superior sensitivity and specificity over conventional fluorescent dyes. Further analyses of the sub-second dynamics of individual nanoparticle extravasation events unveiled distinct characteristics to distinguish between transcellular and paracellular transport. We applied the technique to evaluate the blood-brain barrier (BBB) in zebrafish larvae at different developmental stages, and potential BBB perturbation strategy via nanoparticle functionalisation with polysorbates. Significantly increased BBB penetration by 36.5 folds was shown for the functionalised particles compared to phospholipid-coated particles, attributed to enhanced transcellular crossing. The technique is readily applicable to monitoring vascular integrity and investigating endothelial transport for improved understanding of vascular biology, facilitating advanced research in disease diagnostics and drug delivery towards translation.

biophysics↗

bioRxiv2025

Hybrid Neural-Cognitive Models Reveal Flexible Context-Dependent Information Processing in Reversal Learning

Reversal learning tasks provide a key paradigm for studying behavioral flexibility, requiring individuals to update choices in response to shifting reward contingencies. While reinforcement learning (RL) models have been widely used to provide interpretable explanations of human behavior on similar learning tasks, recent work has revealed that they often fail to fully account for the complexity of learning dynamics. In contrast, artificial neural networks (ANNs) often achieve substantially higher predictive accuracy, but lack the interpretability afforded by classical RL models. To bridge this gap, we introduce HybridRNNs--neural-cognitive models that integrate RL-inspired structures with flexible recurrent architectures. Among them, Context-ANN incorporates latent reward history and choice perseverance, and demonstrates improved alignment with human behavior compared to traditional RL models across two datasets. While not perfectly replicating human strategies, Context-ANN achieves comparable predictive accuracy to generic RNNs and offers interpretable value representations. Additional analyses of hidden dynamics reveal structured, context-sensitive internal states that adapt following reversals. These results suggest that humans may rely on more flexible or context-sensitive learning strategies even in simple reversal tasks, and highlight the potential of HybridRNNs as cognitive models that balance interpretability and predictive accuracy.

animal behavior and cognition↗

bioRxiv2025

Caffeic acid promoted deep vein thrombosis resolution in mice by suppressed macrophage M1 polarization through Keap1/Nrf2 pathwayv

BackgroundDeep vein thrombosis (DVT) carries significant health risks, with macrophages playing a key role in thrombus resolution. Caffeic acid (CA) has been shown to inhibit thrombosis, but its role in DVT resolution remains unclear. PurposeThis study aimed to investigates the effects and mechanisms of CA in accelerating DVT resolution. MethodsA stasis-type DVT model was established in mice via inferior vena cava ligation. The effects of CA were analyzed. Thrombus resolution was assessed using histological staining, gelatin zymography, immunoblotting, and immunofluorescence. Macrophage depletion was conducted with chlorophosphate liposomes, and bone marrow-derived macrophages (BMDMs) were used for polarization studies. RNA sequencing identified potential molecular pathways. Nrf2 gene-deficient mice were used to verify the role of Nrf2 in deep vein thrombus resolution. ResultsCaffeic acid reduced thrombus size, enhanced collagenolysis via increased MMP-2 activity, promoted neovascularization, increased macrophage infiltration, and suppressed M1 polarization and inflammation. CA activated the nuclear factor erythroid 2-related factor 2 (Nrf2) pathway by inhibiting Keap1, leading to enhanced antioxidant responses in both BMDMs and thrombus tissue. Macrophage depletion negated CAs benefits, confirming the central role macrophages. ConclusionCA promoted early thrombus resolution via macrophage recruitment, M1 polarization suppression, and Nrf2 activation, highlighting its potential as a preventive strategy for DVT.

pharmacology and toxicology↗

bioRxiv2025

Signatures of digital Polycomb regulation in functional iPSC heterogeneity between individuals

Induced pluripotent stem cells (iPSCs) show substantial heterogeneity between individuals in their capacity to differentiate into various cell types, hindering their translational application. To explore the causes of this variability, we analysed a panel of ten human iPSC lines, evaluating their differentiation efficiency for two different fates, profiling their transcriptome and chromatin state. Using a machine learning approach, we related chromatin state variability with transcriptional differences. We found that regulation of the Polycomb-mediated histone modification H3K27me3 at specific gene loci exhibited the most consistent differences among iPSC lines, frequently displaying a digital ON/OFF pattern, consistent with a mathematical model based on Polycomb read-write feedback. Notably, altered gene expression patterns of Polycomb-regulated genes were largely propagated into an intermediate state in the differentiation trajectory. Finally, we uncovered a subset of Polycomb-regulated genes with heterogeneous expression and chromatin signatures that were highly predictive of iPSC differentiation efficiencies for two cell types.

systems biology↗

bioRxiv2025

Paraquat Licences α-Synuclein to Trigger NLRP3-Dependent Pyroptosis in Microglia

Parkinsons disease (PD) is characterized by dopaminergic neurodegeneration and the pathological accumulation of -synuclein (Syn) aggregates. While its etiology is multifactorial, environmental toxicants such as paraquat, a widely used herbicide banned in over 70 countries but still permitted in some regions, including Australia, are strongly implicated. We previously showed that Syn activates the NLRP3 inflammasome in microglia, inducing interleukin-1{beta} (IL-1{beta}) release without triggering pyroptosis. Here, we examine how paraquat modulates microglial NLRP3 activation by Syn. Using primary microglia from wild-type (WT) and Nlrp3 knockout (Nlrp3-/-) mice, alongside human monocyte-derived microglia, we demonstrate that paraquat induces dose-dependent NLRP3 activation in LPS-primed WT microglia via NADPH oxidase-mediated reactive oxygen species (ROS) generation. This results in ASC speck formation and IL-1{beta} release, which is absent in Nlrp3-/- microglia and blocked by the selective NLRP3 inhibitor MCC950. While Syn alone fails to induce pyroptosis, co-treatment with paraquat resulted in caspase-1-dependent lactate dehydrogenase (LDH) release and gasdermin-D cleavage, hallmarks of pyroptotic cell death. This response was also suppressed by NLRP3 inhibition. Our findings reveal that paraquat acts as a critical second hit to license Syn-induced inflammasome-dependent pyroptosis in microglia, highlighting a mechanistic link between environmental toxin exposure and innate immune activation in PD pathogenesis. Targeting this axis with brain-penetrant NLRP3 inhibitors may offer an alternative therapeutic avenue for synucleinopathies associated with environmental toxins.

neuroscience↗

bioRxiv2025

Common Signatures of Neutrophils in Diverse Disease Conditions

Neutrophils are traditionally recognized for their pro-inflammatory roles. However, accumulating evidence has begun to highlight the plasticity of transcriptional states of neutrophils during pathological insults. Whether such unconventional neutrophils may share commonality across diverse disease conditions is incompletely understood. Here, we systematically profile over 500,000 neutrophils in key immune compartments and disease-inflicted tissues of the mouse models of metabolic disorder, autoimmunity, tissue damage, peripheral cancers, and intracranial gliomas. Of importance, we observe two distinct neutrophil clusters with unique immune features. The first cluster, characterized by Cd274, Vegfa, and antigen presentation, is highly enriched within the diseased tissues. In contrast, the second cluster with elevated Cd244 and type 2 immune response but reduced maturation markers primarily emerges in the peripheral blood and spleens of specific disease scenarios. These results have elucidated the common signatures of neutrophils in response to various pathological conditions, providing a valuable reference for diagnostic and therapeutic applications.

immunology↗

bioRxiv2025

Accurate and Fast Protein Acylation Identification by Eliminating Position Effects of Cyclic Immonium Ions with Stepped HCD

Mass spectrometry (MS)-based proteomics is indispensable for studying post-translational modifications (PTMs). Cyclic immonium (CycIm) ions serve as invaluable diagnostic markers for lysine acylations, yet the principles governing their generation efficiency are poorly understood. Here, we systematically investigate this question and uncover a robust "position effect": the generation of immonium ions is strongly favored when the modified residue is located near the N-terminus of a tryptic peptide. Utilizing LysargiNase digestion and isotope-labeled synthetic peptides, we demonstrate that this effect is likely driven by the inherent instability of b-type fragment ions during collision-induced dissociation. Furthermore, we show that a stepped HCD strategy enables enhanced sequence coverage and robust CycIm ion detection ([~]99%), thereby improving the depth, reliability and speed of PTM identification. Collectively, this work provides fundamental understanding of immonium ion formation and establishes an optimized acquisition and analysis strategy that enhances the efficiency and confidence of PTM analysis.

biochemistry↗

bioRxiv2025

Electric fields trigger ceramide-dependent budding of exosome vesicles into multivesicular endosomes and boost the generation of exosomes

Exosomes hold immense therapeutic and diagnostic potential, yet their clinical translation remains constrained by low yields. Physiological electric fields (EFs), naturally occurring during wound healing and embryo development, have unexplored roles in exosome biogenesis. Here, we demonstrate that physiological-strength EFs (Direct Current, 50-200 mV/mm) dramatically enhance cellular exosome secretion, achieving a nearly 100-fold increase in a strength-dependent manner. Mechanistically, EFs augmented intraluminal vesicle formation within multivesicular bodies. We proposed a novel EF-driven membrane electroassembly and vesiculogenesis hypothesis and tested it using giant plasma membrane vesicles. We reconstructed EF-induced membrane remodeling and observed EFs driving inward budding of nanoscale exosome-like vesicles. Notably, alternating current EFs (50-400 Hz) exhibited significantly diminished efficacy compared to direct current EFs, highlighting membrane electropolarization-dependent modulation. Pharmacological inhibition of the lipid raft and ceramide almost abolishes EF-induced exosome secretion. Inhibition of the PI3K partially attenuated EF-triggered exosome release. Our findings not only unveil EFs as a potent physiological regulator of exosome secretion but also establish a novel, high-yield production strategy leveraging bioelectric cues. Significant statementWe reported that the bodys natural bioelectricity, the faint electric currents in healing wounds and embryo development, can trigger cells to release 100 times more "natural nanoscale vesicle couriers" called exosomes. This pure physical method is far more efficient than current available technologies, paving the way for breakthroughs in cancer early detection, stem cell therapies, and precision medicine. We found that gentle electric fields can "blow" the cell membrane to release exosomes like "soap bubbles". This explains how bioelectricity controls cell communication during development and wound healing. In short, harnessing the bodys hidden "electric language" unlocks a fast and natural way to mass-produce exosomes.

cell biology↗

bioRxiv2025

An efficient artificial esterase with a dynamic conformation via conformational engineering

The catalytic turnover number (kcat) of current enzyme mimics and de novo designed enzymes is still orders of magnitude lower than that of natural enzymes, because mimicking the fast-dynamic feature of enzyme active centers remains a great challenge. Herein, we created a gold nanoparticle-based artificial esterase, Goldenzyme, by conformational engineering to reconstruct dynamic active centers resembling that of -chymotrypsin. NMR characterization shows that Goldenzyme possesses a fast-dynamic feature, about 6-fold faster than typical -helix. Therefore, Goldenzyme efficiently hydrolyzes p-nitrophenyl acetate with a net kcat of 6.37 s-1 per active center, more than 5-fold of that of -chymotrypsin (1.19 s-1), and Goldenzyme can even hydrolyze some non-activated esters that -chymotrypsin cannot. Our conformational engineering approach demonstrates the possibility of creating artificial enzymes that surpass natural ones.

biochemistry↗

bioRxiv2025

ZCCHC4 Promotes Translation of Replication-dependent Histone mRNAs by Recruiting Cytoplasmic eIF3 complex

Chromatin instability is a major driving force in tumorigenesis, so the correct chromatin structure is essential for maintaining genome stability during the various stages of the cell cycle and DNA template processes. However, there is limited research on how histones are regulated and whether insufficient histone supply leads to chromatin instability, thereby affecting the process of tumor generation. Our study demonstrates that ZCCHC4 exhibits dual subcellular localization in both the nucleus and cytoplasm. Notably, in the cytoplasm, ZCCHC4 facilitates the translation of replication-dependent (RD) histone mRNAs, potentially through direct interaction with components of the translation initiation machinery. Depletion of ZCCHC4 results in a global reduction in protein translation, particularly affecting the translation of replication-dependent histones. This translational impairment prolongs the S phase of the cell cycle, leading to insufficient histone supply, chromatin relaxation, and genomic instability. Moreover, ZCCHC4 knockout markedly inhibits cell proliferation, colony-forming ability, and the growth of xenograft tumors. In conclusion, this study demonstrates that ZCCHC4 plays a pivotal role in regulating global protein translation by interacting with the eIF3 complex, which is essential for efficient mRNA translation in cancer cells and for sustaining the supply of replication-dependent histones during the S phase.

cancer biology↗

bioRxiv2025

KRAS withdrawal in Cholangiocarcinoma leads to immune infiltration and tumor regression

Background and AimsCholangiocarcinoma (CCA) is a liver cancer with poor survival rates. Current treatments, including targeted therapies for specific mutations, are limited and benefit only a small subset of patients. KRAS mutations are found in 15-40% of CCA, representing a new potential treatment target. Whether KRAS inhibition leads to CCA tumor regression is unknown partly due to the lack of conditional animal models. Approach and ResultsWe engineered a conditional KrasG12D-driven CCA mouse model by co-delivering plasmids encoding the Sleeping Beauty transposase with a luciferase reporter, a transposon-borne inducible KrasG12D transgene and Cas9 and Trp53 guide RNA into mouse liver by hydrodynamic tail-vein injection. In vivo bioluminescent imaging showed that KrasG12D withdrawal resulted in 99% tumor regression by day 7. KrasG12D withdrawal resulted in infiltration of activated CD8+ T cells by IHC and IF staining. Single cell RNA-Seq result also validated the enrichment of activated CD8+ T cells subpopulation in KrasG12D-withdrawn tumor. RNA-Seq suggested that KrasG12D withdrawal stimulated transforming growth factor beta pathway and induced senescence. We used cytokine array to characterize the secretion of pro-inflammatory factors, including IL-15 and Ccl17, upon KrasG12D withdrawal. Lentiviral overexpression of murine CCL17 delayed CCA tumor progression in a xenograft model, and overexpression of murine IL-15 resulted in tumor regression in a transplant model. Flow cytometry analysis revealed that IL-15 and CCL17 recruited and activated CD8+ T cells in CCA tumor. Expression of IL-15 resulted in blockade of tumor progression in our TKP CCA model. ConclusionsKrasG12D withdrawal results in rapid tumor regression, highlighting the importance of oncogenic Kras in CCA tumor maintenance. KrasG12D withdrawal induces p53-independent senescence, secretion of pro-inflammatory factors, and immune surveillance by activated CD8+ T cells. We identified two secreted factors IL-15 and CCL17 that could recruit and activate T cells and control CCA tumor progression. This study underscores KRAS inhibition as a potential therapeutic approach for CCA.

cancer biology↗

bioRxiv2025

Epigenetic regulation of cilia stability and kidney by the chromatin remodeling SWI/SNF complexes

Cilia are important subcellular organelles, whose assembly are regulated by master regulator transcription factors including Foxj1 and Rfx proteins. However, whether and how cilia are regulated at epigenetic level remains unknown. We addressed this question by knocking down or knocking out of chromatin remodeling genes. Notably, depletion of multiple components of the switch/sucrose non-fermentable (SWI/SNF) complexes led to ciliopathy-like phenotypes in zebrafish embryos. Specifically, loss of Actl6a, an essential components of the SWI/SNF complexes, resulted in cilia disassembly and cystic kidney, without affectting cilia motility. Omics analyses revealed that in Actl6a-depleted pronephros or embryos, a set of cilia genes--including master regulators foxj1a and rfx2--were downregulated at transcriptional level, chromatin accessibility level and SWI/SNF binding level. Depletion of foxj1a or rfx2 in zebrafish also caused cilia disassembly and cystic kidney. Furthermore, overexpression of foxj1a or rfx2 mRNA partially rescued the cystic kidney and cilia disassembly phenotypes in actl6a mutants. Taken together, our study reveals that the SWI/SNF complexes maintain cilia stability and kidney homeostasis by directly modulating the expression of foxj1a or rfx2.

developmental biology↗

bioRxiv2025

METTL5 Blockade Enhances Anti-Tumor Immune Response via Inducing Neoantigen Generation

Tumor neoantigens play a pivotal role in eliciting tumor-specific immune responses and holds the promise for personalized immunotherapy. However, previous studies mainly focused on the tumor-specific neoantigens derived from genomic mutation and aberrant RNA splicing, limiting the repertoire of targetable neoantigens. Here, we demonstrate that inhibition of rRNA methyltransferase METTL5 translationally increases neoantigen production and enhances anti-tumor immunity. Mechanistically, METTL5-mediated m6A modification at the decoding center of small ribosomal subunit maintains the proper function of ribosome during mRNA translation. METTL5-deficiency decreases translation fidelity and increases production of tumor cell-specific antigens derived from non-canonical translation. Furthermore, we found that Mettl5-depletion increased CD8T cell infiltration density and T cell receptor (TCR) repertoire diversity in murine tumor models. Importantly, this immunostimulatory effect strictly depended on intact antigen presentation pathways, suggesting that Mettl5 knockout exerts its effects primarily through neoantigen generation. Together, this study uncovers the intrinsic mechanisms sustaining mRNA translation accuracy, elucidates a novel source of tumor neoantigen generation, and proposes a new strategy to enhance immunotherapy through targeting mRNA translation.

cancer biology↗

bioRxiv2025

Differentiation status determines tumorigenicity and immunogenicity of cancer cells

Cancer (tumorigenic) cells exhibit cellular properties such as tumorigenicity, evasion of anti-tumor immunity and cell death, metastasis, etc. We showed previously that the core property of cancer cells is neural stemness, which represents general stemness and confers cancer cells with tumorigenicity and pluripotency. Immunogenicity of cancer cells plays the key role in anti-tumor immunity. While tumorigenicity is defined by neural stemness and its regulatory networks, how immunogenicity of cancer cells is determined and how tumorigenicity is related with immunogenicity remained unknown. In the present study, we show that genes conferring tumorigenicity and genes conferring immunogenicity are inversely correlated with differentiation status of cells. Cancer cells can be induced to differentiate into different types of cells either by lineage specific differentiation factors, e.g., muscle differentiation factor MYOD1 or adipocyte differentiation factor PPARG, or by blocking cell-intrinsic oncofactors, e.g., SETDB1. Induced differentiation reprograms both cellular properties and transcriptomes of cancer cells, leading to suppression of genes conferring tumorigenicity and upregulation of genes conferring immunogenicity, and ultimately, leading to loss of neural stemness, suppression of tumorigenicity and enhancement of immunogenicity in cancer cells after differentiation. Vice versa, dedifferentiation leads to the opposite effect. The results identified the cellular mechanism that tumorigenicity and immunogenicity of cancer cells are inversely connected and determined by differentiation status. Such a mechanism, together with our previous studies, reinforces that cancer cell properties should be understood by understanding neural stemness and the principle of embryonic cell/tissue differentiation, and novel cancer therapies can be developed by employing differentiation effect of cancer cells induced by differentiation factors. As a proof of concept test, we showed that muscle differentiation factor MYOD1 suppresses tumorigenesis in a mouse model of hepatocellular carcinoma.

cancer biology↗