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Targeting METTL3 as a checkpoint to enhance T cells for tumour immunotherapy.
BACKGROUND: Immunotherapy has emerged as a crucial treatment modality for solid tumours, yet tumours often evade immune surveillance. There is an imperative to uncover novel immune regulators that can boost tumour immunogenicity and increase the efficacy of immune checkpoint blockade (ICB) therapy. Epigenetic regulators play critical roles in tumour microenvironment remodelling, and N6-methyladenosine (m6A) is known to be involved in tumourigenesis. However, the role of m6A in regulating T-cell function and enhancing anti-tumour immunity remains unexplored. METHODS: Several cancer cell lines were treated with STM2457, an enzymatic inhibitor of RNA m6A methyltransferase METTL3, and explored the transcriptome changes with RNA sequencing (RNA-seq). We then utilised mouse melanoma (B16) and mouse colorectal adenocarcinoma (MC38) models to investigate the effects of METTL3 inhibition on immunotherapy, and analysed the dynamics of the tumour microenvironment via single-cell RNA-seq (scRNA-seq). Furthermore, in vitro and in vivo T-cell cytotoxicity killing assay and CRISPR Cas9-mediated m6A reader YTHDF1-3 knockout in B16 were performed to assess the role and the molecular mechanism of RNA m6A in tumour killing. Finally, the efficacy of METTL3 inhibition was also tested on human melanoma model (A375) and human T cells. RESULTS: We demonstrate that inhibiting METTL3 augments tumour immunogenicity and sustains T-cell function, thereby enhancing responsiveness to ICB therapy. Mechanistically, METTL3 inhibition triggers an interferon response within tumour cells, amplifying the anti-tumour immune response, along with deletion of the m6A reader protein YTHDF2 in tumours inhibiting major histocompatibility complex (MHC)-I degradation. Remarkably, these anti-tumour effects are reliant on the immune system. Specifically, METTL3 inhibition enhances interferon-gamma (IFNγ) and granzyme B (GzmB) expression, thereby strengthening T-cell killing ability, and concurrently dampening the expression of exhaustion-related genes. CONCLUSION: Targeting METTL3 enhances anti-tumour immunity by boosting T-cell cytotoxicity and reversing T-cell exhaustion. Our study positions METTL3 as an epigenetic checkpoint, highlighting the potential of targeting METTL3 to invigorate intrinsic anti-tumour defenses and overcome immune resistance. KEY POINTS: Targeting METTL3 augments tumour cell immunogenicity and sustains T-cell function. T cell with METTL3 inhibition can reverse T-cell exhaustion, and promote expression of IFNγ and GzmB, thereby enhancing cytotoxicity in anti-PD-1 therapy. YTHDF2 deletion in tumours prolong the lifespan of MHC-I mRNAs.
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An integrative single-cell atlas for exploring the cellular and temporal specificity of genes related to neurological disorders during human brain development.
Single-cell technologies have enhanced comprehensive knowledge regarding the human brain by facilitating an extensive transcriptomic census across diverse brain regions. Nevertheless, understanding the cellular and temporal specificity of neurological disorders remains ambiguous due to developmental variations. To address this gap, we illustrated the dynamics of disorder risk gene expression under development by integrating multiple single-cell RNA sequencing datasets. We constructed a comprehensive single-cell atlas of the developing human brain, encompassing 393,060 single cells across diverse developmental stages. Temporal analysis revealed the distinct expression patterns of disorder risk genes, including those associated with autism, highlighting their temporal regulation in different neuronal and glial lineages. We identified distinct neuronal lineages that diverged across developmental stages, each exhibiting temporal-specific expression patterns of disorder-related genes. Lineages of nonneuronal cells determined by molecular profiles also showed temporal-specific expression, indicating a link between cellular maturation and the risk of disorder. Furthermore, we explored the regulatory mechanisms involved in early brain development, revealing enriched patterns of fetal cell types associated with neuronal disorders indicative of the prenatal stage's influence on disease determination. Our findings facilitate unbiased comparisons of cell type‒disorder associations and provide insight into dynamic alterations in risk genes during development, paving the way for a deeper understanding of neurological disorders.
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A State-Resolved Single-Cell and Spatial Atlas Reveals IDH-Associated Differences in Malignant and Immune Architecture in Diffuse Glioma.
Adult-type diffuse gliomas are defined by the isocitrate-dehydrogenase (IDH) axis, yet how this biology constrains malignant cell-state repertoires and their immune-modulatory potential remains unclear. We profiled 69 tumors using single-cell RNA sequencing (scRNA-seq), single-nucleus RNA sequencing (snRNA-seq), and spatial transcriptomics, and resolved seven recurrent malignant cell states: astrocyte-like progenitor cells (ALPC), hypoxia, cycling G1/S, cycling G2/M, oligo-lineage, neuro-oligo-lineage, and neuro-lineage. The IDH axis strongly biased state occupancy and malignant-immune composition, with IDH-mutant tumors dominated by differentiated lineage-like states and reduced immune infiltration, whereas glioblastoma (GBM) showed expansion of ALPC and hypoxia states alongside increased myeloid and lymphoid infiltration. Orthogonal analyses supported that ALPC-associated markers are expressed in situ and in patient-derived stem cell-permissive cultures, consistent with a stem-like, differentiation-competent malignant phenotype. State-resolved pathway analysis identified mitochondrial oxidative phosphorylation programs enriched in GBM ALPC and extracellular matrix remodeling, and chemotaxis-associated programs enriched in GBM hypoxia. Composition-controlled, patient-level interaction modeling identified a greater malignant-myeloid signaling interface in GBM, driven by SPP1-CD44/integrin signaling and confirmed by differential expression; GBM networks also featured TGFB and NECTIN modules, whereas IDH-mutant networks were dominated by APP/APOE-TREM2/TYROBP and MIF modules. These data suggest that the IDH axis shapes glioma behavior by constraining malignant state repertoires and biasing the deployment of state-specific metabolic and immunomodulatory programs.
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A Single-Cell Atlas of Transcriptome Changes in the Intestinal Epithelium at the Suckling-to-Weaning Transition in Male Rabbits.
BACKGROUND & AIMS: The suckling-to-weaning dietary transition is a key step in intestinal development. The aim of our study was to identify the transcriptome changes induced in each cell type of the intestinal epithelium at the onset of solid food ingestion. METHODS: We compared the single-cell transcriptome of epithelial cells isolated from the cecum of age-matched littermate suckling male rabbits ingesting or not solid food. RESULTS: Our dataset provides the first single-cell atlas of the rabbit intestinal epithelium and highlights the interest of the rabbit as a model for studying BEST4+ epithelial cells, which are absent in mice. Solid food ingestion induced extensive transcriptome changes in each epithelial cell type, with the most pronounced changes noted in absorptive and BEST4+ cells. Some of the effects of solid food introduction were common to most epithelial cell types, such as the upregulation of ALDH1A1. Solid food ingestion remodeled epithelial defense systems, as observed by the increased expression of interferon-stimulated genes in mature absorptive and BEST4+ cells. Solid food upregulated the gene expression of the immunoglobulin transporter PIGR in cells located at the base of epithelial crypts and in goblet cells. In addition, solid food triggered epithelial differentiation, which was associated with modification of the expression of genes involved in handling of nutrients, as well as changes in hormone expression by enteroendocrine cells. These cell type-specific transcriptome modifications induced by solid food ingestion coincided with changes in microbiota composition and were replicated, in part, by butyrate in organoids. CONCLUSIONS: Our work provides a single-cell atlas of the transcriptome changes induced in the intestinal epithelium at the suckling-to-weaning transition.
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A new atlas to study embryonic cell types in Xenopus.
This paper introduces a single-cell atlas for pivotal developmental stages in Xenopus, encompassing gastrulation, neurulation, and early tailbud. Notably surpassing its predecessors, the new atlas enhances gene mapping, read counts, and gene/cell type nomenclature. Leveraging the latest Xenopus tropicalis genome version, alongside advanced alignment pipelines and machine learning for cell type assignment, this release maintains consistency with previous cell type annotations while rectifying nomenclature issues. Employing an unbiased approach for cell type assignment proves especially apt for embryonic contexts, given the considerable number of non-terminally differentiated cell types. An alternative cell type attribution here adopts a fuzzy, non-deterministic stance, capturing the transient nature of early embryo progenitor cells by presenting an ensemble of types in superposition. The value of the new resource is emphasized through numerous examples, with a focus on previously unexplored germ cell populations where we uncover novel transcription onset features. Offering interactive exploration via a user-friendly web portal and facilitating complete data downloads, this atlas serves as a comprehensive and accessible reference.
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AJUBA: The Master Regulator Bridging EMT and Immune Evasion in Colorectal Cancer.
BACKGROUND: Epithelial-mesenchymal transition (EMT) represents a critical process that facilitates metastatic dissemination and immune evasion in colorectal cancer (CRC); however, the molecular factors that connect EMT to modifications in the immune microenvironment remain poorly elucidated. In this investigation, we identify AJUBA as an essential regulator that mediates the association between EMT and immune modulation in CRC. METHODS: By integrating multi-cohort transcriptomic datasets (The Cancer Genome Atlas (TCGA)-CRC, GSE18105, GSE22598, GSE89076, and GSE110224) with single-cell RNA-seq data (GSE132465), we applied machine learning and deep learning methodologies to comprehensively identify EMT-associated genes demonstrating prognostic significance. AJUBA validation was performed at mRNA and protein levels in a cohort of 90 CRC patient samples using quantitative PCR, Western blotting, and immunohistochemical (IHC) approaches. Functional analyses involved siRNA-mediated knockdown experiments, coupled with evaluations of cell proliferation (CCK-8 assay), migration and invasion (transwell assay), clonogenic capacity (colony formation assay), and in vivo tumor growth in xenograft models. Immune infiltration was assessed via ssGSEA and CIBERSORT algorithms, and spatial transcriptomics data (GSE225857) were used to delineate AJUBA expression within tumor microdomains. RESULTS: Across multiple CRC cohorts, AJUBA exhibited marked upregulation and showed distinct enrichment in epithelial cells with activated EMT characteristics. Spatial transcriptomic profiling demonstrated AJUBA colocalization with cancer-associated fibroblasts (CAFs) within immune-excluded niches. Enhanced AJUBA expression exhibited a positive correlation with heightened infiltration of M2 macrophages and activation of VEGF/NOTCH signaling cascades. In vivo, AJUBA knockdown led to suppressed tumor growth, reduced Ki-67 proliferation indices, and diminished M2 macrophage abundance. Clinically, elevated AJUBA expression correlated with advanced nodal metastasis and served as an independent predictor of poor overall survival (OS; HR = 4.809, 95% CI: 2.385-9.695, p < 0.001). CONCLUSIONS: AJUBA functions as a key regulator that links EMT to immune modulation, promoting macrophage polarization and facilitating immune evasion in CRC. Through its coupling of EMT activation with proangiogenic signaling, AJUBA represents both a prognostic biomarker and a promising therapeutic target for alleviating immune exclusion in metastatic CRC.
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Single-cell and bulk RNA sequencing reveal cancer-associated fibroblast heterogeneity and a prognostic signature in prostate cancer.
Cancer-associated fibroblasts (CAFs), the central players in the tumor microenvironment (TME), can promote tumor progression and metastasis via various functions. However, the properties of CAFs in prostate cancer (PCa) have not been fully assessed. Therefore, we aimed to examine the CAF characteristics in PCa and construct a CAF-derived signature to predict PCa prognosis. CAFs were identified using single-cell RNA sequencing (scRNA-seq) data from 3 studies. We performed the FindAllMarkers function to extract CAF marker genes and constructed a signature to predict the biochemical relapse-free survival (bRFS) of PCa in the Cancer Genome Atlas (TCGA) cohort. Subsequently, different algorithms were applied to reveal the differences of the TME, immune infiltration, treatment responses in the high- and low-risk groups. Additionally, the CAF heterogeneity was assessed in PCa, which were confirmed by the functional enrichment analysis, gene set enrichment analysis (GSEA), and AUCell method. The scRNA-seq analysis identified a CAF cluster with 783 cells and determined 183 CAF marker genes. Cell-cell communication revealed extensive interactions between fibroblasts and immune cells. A CAF-related prognostic model, containing 7 genes (ASPN, AEBP1, ALDH1A1, BGN, COL1A1, PAGE4 and RASD1), was developed to predict bRFS and validated by 4 independent bulk RNA-seq cohorts. Moreover, the high-risk group of the signature score connected with an immunosuppressive TME, such as a higher level of M2 macrophages and lower levels of plasma cells and CD8+ T cells, and a reduced reaction rate for immunotherapy compared with low-risk group. After re-clustering CAFs via unsupervised clustering, we revealed 3 biologically distinct CAF subsets, namely myofibroblast-like CAFs (myCAFs), immune and inflammatory CAFs (iCAFs) and antigen-presenting CAFs (apCAFs). In conclusion, the CAF-derived signature, the first of its kind, can effectively predict PCa prognosis and serve as an indicator for immunotherapy. Furthermore, our study identified 3 CAF subpopulations with distinct functions in PCa.
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Identification of CXCL12 as a key gene of trophoblast and endometrial stromal cell decidualization in pregnancy via scRNA-seq and experimental validation.
BACKGROUND: Recurrent miscarriage (RM) remains a significant clinical challenge due to its elusive pathogenesis. This study aimed to investigate the role of chemokine ligand 12 (CXCL12) at the maternal-fetal interface and its specific mechanism in RM, to provide an experimental basis for potential diagnostic and therapeutic strategies. METHODS: We utilized single-cell RNA-sequencing (scRNA-seq) to distinct single-cell atlas at the maternal fetal interface in normal and RM samples. And we comparably analyzed the expression of CXCL12 of Syncytiotrophoblast/Extravillous Trophoblast (SCT/EVET) in normal and unexplained RM pregnancies at the early stage of gestation. A CXCL12 silencing and overexpression system was established in the HTR-8/SVneo cell line. Cell viability was determined by MTT assay. The horizontal migration ability of HTR-8 cells was determined by cell scratch assay, while the invasion ability was tested by transwell matrix gel invasion assay. Recombinant human CXCL12 was used to stimulate endometrial stromal cells to explore the role of CXCL12 in the process of endometrial decidualization. RESULTS: Single-cell atlas at the maternal fetal interface in normal and RM samples showed 11 cell types, including DSCs and SCT/EVT cells. In SCT/EVT cells, CXCL12 was remarkably decreased in RM cases. CXCL12 overexpression significantly promoted the proliferation, migration, and invasion ability of HTR-8 cells, while silencing CXCL12 significantly inhibited the proliferation, migration, and invasion ability of HTR-8 cells. Recombinant human CXCL12 significantly promoted the decidualization process of endometrial stromal cells, while decidualization was inhibited by CXCR4-neutralizing antibodies to a certain extent. CONCLUSION: CXCL12 promotes the proliferation, migration, and invasion ability of trophoblast cells in vitro and promotes the decidualization of endometrial stromal cells. The downregulation of the CXCL12/CXCR4 axis may be involved in the occurrence and development of RM. Thus, this study provides experimental clues for exploring potential therapeutic approaches related to CXCL12.
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Insights into early-onset colorectal cancer from a single-nucleus RNA-seq atlas of colorectal cancer across the age continuum.
The incidence of early-onset colorectal cancer (EO-CRC; diagnosed before age 50 years) is increasing worldwide. Although current data indicate that EO-CRC may be biologically distinct from average-onset CRC (AO-CRC; diagnosed at age ≥50 years), molecular differences remain poorly defined. To address this knowledge gap, we generated a single-nucleus RNA sequencing (snRNA-seq) atlas from 189 primary CRC tumors (54 EO-CRC, 135 AO-CRC) spanning eight decades of life, with at least 20 tumors in most age decades. Our analyses stratified tumors by microsatellite status and accounted for tumor sidedness, stage, patient sex and neoadjuvant treatment history, modeling age continuously or categorically (EO-CRC versus AO-CRC). Among microsatellite-stable (MSS) tumors, those from younger patients were characterized by increased stromal and decreased epithelial proportions, which we confirmed using multispectral immunofluorescence and machine learning in an external cohort of 712 MSS CRC tumors (18 EO-CRC, 694 AO-CRC). Tumor epithelial cells from young patients exhibited increased expression of long noncoding RNAs and FN1, with a trend toward increased heterogeneity in copy number alterations. MSS left-sided EO-CRC tumors had more cell-cell interactions, with epithelial-to-mesenchymal transition-related signaling pathways (FN1/GDF/ACTIVIN) showing increased, cell type-specific interactions. Our large high-resolution snRNA-seq atlas uncovers age-associated features of CRC and provides a resource for the community to understand EO-CRC biology.
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The Immune Atlas of Human Deciduas With Unexplained Recurrent Pregnancy Loss.
Recurrent pregnancy loss (RPL) is a common fertility problem that affects 1%-2% of couples all over the world. Despite exciting discoveries regarding the important roles of the decidual natural killer cell (dNK) and regulatory T cell in pregnancy, the immune heterogeneity in patients with unexplained recurrent pregnancy loss (URPL) remains elusive. Here, we profiled the transcriptomes of 13,953 CD45+ cells from three normal and three URPL deciduas. Based on our data, the cellular composition revealed three major populations of immune cells including dNK cell, T cell, and macrophage, and four minor populations including monocytes, dendritic cell (DC), mast cell, and B cell. Especially, we identified a subpopulation of CSF1+ CD59+ KIRs-expressing dNK cells in normal deciduas, while the proportion of this subpopulation was decreased in URPL deciduas. We also identified a small subpopulation of activated dDCs that were accumulated mainly in URPL deciduas. Furthermore, our data revealed that in decidua at early pregnancy, CD8+ T cells exhibited cytotoxic properties. The decidual macrophages expressed high levels of both M1 and M2 feature genes, which made them unique to the conventional M1/M2 classification. Our single-cell data revealed the immune heterogeneity in decidua and the potentially pathogenic immune variations in URPL.