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Immunodynamic axis of fibroblast-driven neutrophil infiltration in acute pancreatitis: NF-κB-HIF-1α-CXCL1.
BACKGROUND: Acute pancreatitis (AP) is a sterile inflammation, and 10-20% of cases can progress to severe acute pancreatitis (SAP), which seriously threatens human life and health. Neutrophils and their extracellular traps (NETs) play an important role in the progression of AP. However, the immunodynamic factors between the excessive infiltration of neutrophils during the occurrence of AP have not been fully elucidated. METHODS: Adult male C57BL/6 J mice were selected. An AP model was induced by cerulein, and a control group was set up. Single-cell sequencing technology was used to reveal the cell atlas of AP pancreatitis tissue. In vivo, the model mice were treated with anti-Ly6G antibody, DNase I, SC75741, PX-478, and SRT3109 respectively. In vitro, human pancreatic stellate cells were treated with hypoxia, H2O2, NAC, and JSH-2, and co-cultured with neutrophils in Transwell chambers. The severity of inflammation was evaluated, and the molecular mechanism by which fibroblasts exacerbate AP was revealed through techniques such as cell colony formation assay, cell migration assay, cell transfection, immunofluorescence, flow cytometry, Western blot, reverse-transcription quantitative polymerase chain reaction (RT-qPCR), and co-immunoprecipitation (co-IP). RESULTS: The study showed that the elimination of neutrophils and NETs could significantly improve AP. Single-cell RNA sequencing (scRNA-seq) indicated that both neutrophils and fibroblasts in pancreatic tissue exhibited heterogeneity during AP. Among them, neutrophils highly expressed CXCR2, and fibroblasts highly expressed CXCL1. Further experimental results demonstrated that the infiltration of neutrophils in the early stage of AP was related to the activation of fibroblasts. The activation of fibroblasts depended on the nuclear factor kappa B (NF-κB) signaling pathway induced by hypoxia. NF-κB enhanced the activation of pancreatic stellate cells (PSCs) and the secretion of CXCL1 by directly promoting the transcription of HIF-1α and indirectly inhibiting PHD2, resulting in the accumulation of HIF-1α protein. The NF-κB-HIF-1α signal promoted the secretion of CXCL1 by fibroblasts through glycolysis and induced the infiltration of neutrophils. Finally, blocking the NF-κB-HIF-1α-CXCL1 signaling axis in vivo reduced the infiltration of neutrophils and improved AP. CONCLUSIONS: This study, for the first time, demonstrated that activation of fibroblasts is one of the immunological driving factors for neutrophil infiltration and elucidated that glycolysis driven by the NF-κB-HIF-1α pathway is the intrinsic molecular mechanism by which fibroblasts secrete CXCL1 to chemotactically attract neutrophils. This finding provides a highly promising target for the treatment of AP.
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The Dynamically Evolving Cell States and Ecosystem from Benign Nevi to Melanoma.
PURPOSE: Approximately 30% of nonchronically sun-damaged melanomas originate from nevi, yet the dynamic changes and crucial mechanisms driving the transition from benign nevi to melanoma remain elusive. EXPERIMENTAL DESIGN: In this study, we performed single-cell transcriptome sequencing on multiple paired tissue sites from five patients diagnosed with melanoma arising in congenital melanocytic nevi, identifying four distinct states of melanocyte subpopulations during the progression from nevi to melanoma, characterized by dynamic changes in their functions and regulatory pathways. RESULTS: In the nevi state, IFN regulatory factor 1 was specifically upregulated in melanocytes, fibroblasts, and endothelial cells, potentially activating immune surveillance in the microenvironment. Conversely, the critical inhibitory checkpoint HLA-E for NK cells exhibited high expression in a cluster of malignant melanocytes and fibroblasts enriched in melanoma. This interaction with ligands expressed in NK cells could potentially serve as a key factor, leading to immune evasion. In malignant melanoma samples, we detected high expression of midkine in melanocytes. It is a pivotal factor that facilitates melanoma invasion and malignant transformation, potentially through interaction with endothelial cells to stimulate angiogenesis. The targets identified in our study are crucial factors in detecting the malignant transformation of nevi. Ultimately, we developed a malignant progression model capable of predicting patient prognosis and malignant progression status using bulk RNA sequencing data. CONCLUSIONS: Our study provides a high-resolution atlas of the malignant transformation of melanoma from nevi and highlights potential targets for further investigation.
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Multi‑omics identification of a novel signature for serous ovarian carcinoma in the context of 3P medicine and based on twelve programmed cell death patterns: a multi-cohort machine learning study.
BACKGROUND: Predictive, preventive, and personalized medicine (PPPM/3PM) is a strategy aimed at improving the prognosis of cancer, and programmed cell death (PCD) is increasingly recognized as a potential target in cancer therapy and prognosis. However, a PCD-based predictive model for serous ovarian carcinoma (SOC) is lacking. In the present study, we aimed to establish a cell death index (CDI)-based model using PCD-related genes. METHODS: We included 1254 genes from 12 PCD patterns in our analysis. Differentially expressed genes (DEGs) from the Cancer Genome Atlas (TCGA) and Genotype-Tissue Expression (GTEx) were screened. Subsequently, 14 PCD-related genes were included in the PCD-gene-based CDI model. Genomics, single-cell transcriptomes, bulk transcriptomes, spatial transcriptomes, and clinical information from TCGA-OV, GSE26193, GSE63885, and GSE140082 were collected and analyzed to verify the prediction model. RESULTS: The CDI was recognized as an independent prognostic risk factor for patients with SOC. Patients with SOC and a high CDI had lower survival rates and poorer prognoses than those with a low CDI. Specific clinical parameters and the CDI were combined to establish a nomogram that accurately assessed patient survival. We used the PCD-genes model to observe differences between high and low CDI groups. The results showed that patients with SOC and a high CDI showed immunosuppression and hardly benefited from immunotherapy; therefore, trametinib_1372 and BMS-754807 may be potential therapeutic agents for these patients. CONCLUSIONS: The CDI-based model, which was established using 14 PCD-related genes, accurately predicted the tumor microenvironment, immunotherapy response, and drug sensitivity of patients with SOC. Thus this model may help improve the diagnostic and therapeutic efficacy of PPPM.
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Denosumab Enhances Antitumor Immunity by Suppressing SPP1 and Boosting Cytotoxic T Cells.
Denosumab, a RANK ligand inhibitor, is primarily used to prevent osteoclastogenesis in the treatment of conditions such as osteoporosis, bone metastasis, and giant cell tumor of bone (GCTB). RANK ligand also plays an important role in immunity by activating NF-κB and its target genes, including the osteopontin-coding gene SPP1 (also known as OPN), which is linked to CXCL9:SPP1 macrophage polarization and prognosis. In this study, we explored an additional role of denosumab in enhancing antitumor immunity in patients. Single-cell RNA sequencing was performed on nine human GCTB samples, including six untreated and three treated only with denosumab, to exclude confounding treatment factors linked with bone metastasis samples. We further analyzed paired samples collected before and after denosumab treatment from a cohort of nine patients with GCTB and conducted a pan-cancer analysis of 34 distinct types of cancers. Our single-cell analysis of GCTB resulted in a comprehensive cell atlas revealing an antitumor role of denosumab in inhibiting SPP1 expression and augmenting active cytotoxic T-cell abundance. Furthermore, we validated this immunomodulatory role of denosumab using the paired GCTB samples. Finally, the pan-cancer analysis supported a negative correlation between SPP1 and CD8A levels, with the CD8A:SPP1 ratio correlating with overall survival in 14 cancer types, which was superior to either CD8A or SPP1 alone. Our research provides clinical evidence that denosumab improves antitumor immunity by decreasing SPP1 expression and enhancing cytotoxic T-cell activity, serving as a milestone in the development of innovative use of denosumab and offering potential benefits to patients with elevated levels of SPP1.
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Human Body Single-Cell Atlas of 3D Genome Organization and DNA Methylation.
Higher-order chromatin structure and DNA methylation are critical for gene regulation, but how these vary across the human body remains unclear. We performed multi-omic profiling of 3D genome structure and DNA methylation for 86,689 single nuclei across 16 human tissues, identifying 35 major and 206 cell subtypes. We revealed extensive changes in CG and non-CG methylation across almost all cell types and characterized 3D chromatin structure at an unprecedented cellular resolution. Intriguingly, extensive discrepancies exist between cell types delineated by DNA methylation and genome structure, indicating that the role of distinct epigenomic features in maintaining cell identity may vary by lineage. This study expands our understanding of the diversity of DNA methylation and chromatin structure and offers an extensive reference for exploring gene regulation in human health and disease.
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Renal compartment-specific genetic variation analyses identify new pathways in chronic kidney disease.
Chronic kidney disease (CKD), a condition in which the kidneys are unable to clear waste products, affects 700 million people globally. Genome-wide association studies (GWASs) have identified sequence variants for CKD; however, the biological basis of these GWAS results remains poorly understood. To address this issue, we created an expression quantitative trait loci (eQTL) atlas for the glomerular and tubular compartments of the human kidney. Through integrating the CKD GWAS with eQTL, single-cell RNA sequencing and regulatory region maps, we identified novel genes for CKD. Putative causal genes were enriched for proximal tubule expression and endolysosomal function, where DAB2, an adaptor protein in the TGF-β pathway, formed a central node. Functional experiments confirmed that reducing Dab2 expression in renal tubules protected mice from CKD. In conclusion, compartment-specific eQTL analysis is an important avenue for the identification of novel genes and cellular pathways involved in CKD development and thus potential new opportunities for its treatment.
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Tie2 activity in cancer associated myofibroblasts serves as novel target against reprogramming of cancer cells to embryonic-like cell state and associated poor prognosis in oral carcinoma patients.
BACKGROUND: Myofibroblastic cancer-associated fibroblasts (CAF) in tumor stroma serves as an independent poor prognostic indicator, supporting higher stemness in oral cancer; however, the underlying biology is not fully comprehended. Here, we have explored the crucial role of Tunica Interna Endothelial Cell Kinase (Tie2/TEK) signaling in transition and maintenance of myofibroblastic phenotype of CAFs, and as possible link with the poor prognosis of head and neck squamous cell carcinoma (HNSCC) patients. METHODS: Bulk and single cell RNA-sequencing (scRNAseq) methods and in-depth bioinformatic analysis were applied for CAF and cancer cells co-culture for studying molecular relationships. In vitro 3D-spheroid-forming ability, expression of stemness markers, in vivo tumor formation ability in zebrafish embryo and syngeneic mouse allografts formation was conducted to test stemness, upon targeting CAF-specific Tie2 activity by gene silencing or with small molecule inhibitor. Immunohistochemistry analysis was performed to locate the distribution of Tie2 and αSMA in primary tumors of oral carcinoma. Prognosis in HNSCC patient cohort from The Cancer Genome Atlas (TCGA) study was analysed based on single sample gene set enrichment score (ssGSEA) and Kaplan-Meier analysis. RESULTS: Autocrine or exogenous TGFβ-induction in CAF led to the recruitment of histone deacetylase 2 (HDAC2) on the promoter of Tie2-antagonist, Angiopoietin-2 (ANGPT2), resulting in its downregulation, leading to phosphorylation of Tie2 (Y992) and subsequent activation of SRC (Y418). This led to SRC/ROCK mediated αSMA-positive stress-fiber formation with gain of myofibroblast phenotype. The CAF-specific Tie2-signaling was responsible for producing embryonic-like cell state in co-cultured cancer cells; with enhanced tumor initiating ability. Tie2 activity in CAF exerted the dynamic gene expression reprogramming, with the upregulation of 'cell migration' and downregulation of 'protein biosynthesis' related gene-regulatory-network modules in malignant cells. The AUCell scores calculated for gene signatures derived from these modules showed significant concordance in independently reported scRNAseq studies of HNSCC tumors and significant association with poor prognosis in HNSCC patient cohort. CONCLUSIONS: CAF-specific Tie2 activity may serve as direct stromal-target against cancer cell plasticity leading to poor prognosis of oral cancer patients. Overall, our work has provided wider applicability of Tie2-specific functions in tumor biology, along with its known role in endothelial cell-specific function.
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Nicotinamide-N-methyltransferase is a promising metabolic drug target for primary and metastatic clear cell renal cell carcinoma.
BACKGROUND: The metabolic enzyme nicotinamide-N-methyltransferase (NNMT) is highly expressed in various cancer entities, suggesting tumour-promoting functions. We systematically investigated NNMT expression and its metabolic interactions in clear cell renal cell carcinoma (ccRCC), a prominent RCC subtype with metabolic alterations, to elucidate its role as a drug target. METHODS: NNMT expression was assessed in primary ccRCC (n = 134), non-tumour tissue and ccRCC-derived metastases (n = 145) by microarray analysis and/or immunohistochemistry. Findings were validated in The Cancer Genome Atlas (kidney renal clear cell carcinoma [KIRC], n = 452) and by single-cell analysis. Expression was correlated with clinicopathological data and survival. Metabolic alterations in NNMT-depleted cells were assessed by nontargeted/targeted metabolomics and extracellular flux analysis. The NNMT inhibitor (NNMTi) alone and in combination with the inhibitor 2-deoxy-D-glucose for glycolysis and BPTES (bis-2-(5-phenylacetamido-1,3,4-thiadiazol-2-yl)ethyl-sulfide) for glutamine metabolism was investigated in RCC cell lines (786-O, A498) and in two 2D ccRCC-derived primary cultures and three 3D ccRCC air-liquid interface models. RESULTS: NNMT protein was overexpressed in primary ccRCC (p = 1.32 × 10-16 ) and ccRCC-derived metastases (p = 3.92 × 10-20 ), irrespective of metastatic location, versus non-tumour tissue. Single-cell data showed predominant NNMT expression in ccRCC and not in the tumour microenvironment. High NNMT expression in primary ccRCC correlated with worse survival in independent cohorts (primary RCC-hazard ratio [HR] = 4.3, 95% confidence interval [CI]: 1.5-12.4; KIRC-HR = 3.3, 95% CI: 2.0-5.4). NNMT depletion leads to intracellular glutamine accumulation, with negative effects on mitochondrial function and cell survival, while not affecting glycolysis or glutathione metabolism. At the gene level, NNMT-depleted cells upregulate glycolysis, oxidative phosphorylation and apoptosis pathways. NNMTi alone or in combination with 2-deoxy-D-glucose and BPTES resulted in inhibition of cell viability in ccRCC cell lines and primary tumour and metastasis-derived models. In two out of three patient-derived ccRCC air-liquid interface models, NNMTi treatment induced cytotoxicity. CONCLUSIONS: Since efficient glutamine utilisation, which is essential for ccRCC tumours, depends on NNMT, small-molecule NNMT inhibitors provide a novel therapeutic strategy for ccRCC and act as sensitizers for combination therapies.
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Prion protein regulates invasiveness in glioblastoma stem cells.
BACKGROUND: Glioblastoma (GBM) is an aggressive brain tumor driven by glioblastoma stem cells (GSCs), which represent an appealing target for therapeutic interventions. The cellular prion protein (PrPC), a scaffold protein involved in diverse cellular processes, interacts with various membrane and extracellular matrix molecules, influencing tumor biology. Herein, we investigate the impact of PrPC expression on GBM. METHODS: To address this goal, we employed CRISPR-Cas9 technology to generate PrPC knockout (KO) glioblastoma cell lines, enabling detailed loss-of-function studies. Bulk RNA sequencing followed by differentially expressed gene and pathway enrichment analyses between U87 or U251 PrPC-wild-type (WT) cells and PrPC-knockout (KO) cells were used to identify pathways regulated by PrPC. Immunofluorescence assays were used to evaluate cellular morphology and protein distribution. For assessment of protein levels, Western blot and flow cytometry assays were employed. Transwell and growth curve assays were used to determine the impact of loss-of-PrPC in GBM invasiveness and proliferation, respectively. Single-cell RNA sequencing analysis of data from patient tumors from The Cancer Genome Atlas (TCGA) and the Broad Institute of Single-Cell Data Portal were used to evaluate the correspondence between our in vitro results and patient samples. RESULTS: Transcriptome analysis of PrPC-KO GBM cell lines revealed altered expression of genes associated with crucial tumor progression pathways, including migration, proliferation, and stemness. These findings were corroborated by assays that revealed impaired invasion, migration, proliferation, and self-renewal in PrPC-KO GBM cells, highlighting its critical role in sustaining tumor growth. Notably, loss-of-PrPC disrupted the expression and localization of key stemness markers, particularly CD44. Additionally, the modulation of PrPC levels through CD44 overexpression further emphasizes their regulatory role in these processes. CONCLUSIONS: These findings establish PrPC as a modulator of essential molecules on the cell surface of GSCs, highlighting its potential as a therapeutic target for GBM.
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Multi-omics analysis identifies CCNB1 as a cell cycle factor driving glioblastoma progression and its inhibition by resveratrol.
Glioblastoma (GBM) is a fast-growing primary brain tumor with high mortality and recurrence rates. Dysregulation of the cell cycle is a hallmark of GBM, and cyclin B1 (CCNB1) is a key regulator of the cell cycle. However, the role of CCNB1 in GBM remains unclear. In this study, we found that CCNB1 mRNA and protein expression levels were significantly higher in GBM tissues than normal tissues. High CCNB1 mRNA expression was associated with poorer prognosis in GBM patients. Single-cell and spatial transcriptomics data revealed that CCNB1+ cells represent a proliferative subcluster in GBM, annotated as proliferative cells, and characterized by the upregulation of cell cycle-related pathways. CCNB1 inhibition decreased the proliferation of GBM cells and impaired cell cycle progression from S phase to G2/M. Additionally, resveratrol could inhibit the expression of CCNB1 and its interacting gene polo-like kinase 1 (PLK1). Importantly, through in vitro and in vivo experiments, we found that resveratrol suppressed GBM cell growth with low toxicity. CCNB1 silencing combined with resveratrol treatment further inhibited the proliferation of GBM cells. Collectively, these data suggest that CCNB1 is highly expressed in GBM and may promote GBM progression. Inhibition of CCNB1 may represent a potential therapeutic strategy for GBM.