Single-cell RNA Sequencing Analysis Research Articles

424 (PB412): Molecular drivers of plasticity and immunosuppression in a single-cell RNA sequencing and multicolor image analysis of human small-cell lung cancer

424 (PB412): Molecular drivers of plasticity and immunosuppression in a single-cell RNA sequencing and multicolor image analysis of human small-cell lung cancer

P53 mutation biases squamocolumnar junction progenitor cells towards dysplasia rather than metaplasia in Barrett’s oesophagus

BackgroundWhile p53 mutations occur early in Barrett’s oesophagus (BE) progression to oesophageal adenocarcinoma (EAC), their role in gastric cardia stem cells remains unclear.ObjectiveThis study investigates the impact of p53 mutation...

The critical involvement of monocytes/macrophages in the pathogenesis of primary Sjögren's syndrome: New evidence from Mendelian randomization and single-cell sequencing

BackgroundPrimary Sjögren's syndrome (pSS) stands as a chronic autoimmune disease characterized by an elusive pathogenesis. The synergy of single-cell RNA sequencing and Mendelian randomization (MR) analysis provides an opportunity to comprehensively unravel the contributory role of monocytes/macrophages in the intricate pathogenesis of pSS. MethodsDifferentially expressed genes (DEGs) of various types of immune cells were analyzed after annotating single-cell RNA sequencing (scRNA-seq) data. MR analysis of expression quantitative trait loci (eQTL) and protein quantitative trait loci (pQTL) was conducted to search for key pathogenic genes and proteins. Cellular localization of pathogenic genes was performed based on scRNA-seq data. Variations in signaling pathways between immune cells were further analyzed. ResultsA total of 1434 significant DEGs were identified. Among these, 60 genes exhibited strong relevance to the occurrence of pSS, of which 32 genes differentially expressed in monocytes/macrophages. CTSS was found to be a significant risk protein with a p-value of 0.001 and an odds ratio of 1.384 (1.147–1.669), showing pronounced expression in monocytes/macrophages. Furthermore, monocytes/macrophages displayed heightened expression levels of MXD1, AMPD2, TNFSF10, FTL, UBXN11, CSF3R, and LILRA5. The analysis of intercellular signaling revealed increased signal intensity in both incoming and outgoing signals in monocytes/macrophages. The signaling interactions between monocytes/macrophages, B cells, and T cells exhibited varying degrees of deviation. ConclusionsThis study highlights the significant involvement of monocytes/macrophages in the pathogenesis of pSS, as evidenced by MR analysis and scRNA-seq analysis. This suggests monocytes/macrophages as a focal point for pathogenesis research and potential therapeutic targeting in pSS.

Exercise Induced Endothelial Mesenchymal Transition (EndMT) Facilitates Meniscal Fibrocartilage Regeneration.

The meniscus is a semilunar wedge-shaped fibrocartilage tissue within the knee joint that is important for withstanding mechanical shock during joint motion. The intrinsic healing capacity of meniscus tissue is very limited, which makes meniscectomy the primary treatment method in the clinic. An effective translational strategy for regenerating the meniscus after total or subtotal meniscectomy, particularly for extensive meniscal lesions or degeneration, is yet to be developed. The present study demonstrates that the endothelial mesenchymal transition (EndMT) contributes to meniscal regeneration. The mechanical stimulus facilitated EndMT by activating TGF-β2 signaling. A handheld bioprinter system to intraoperatively fabricate a porous meniscus scaffold according to the resected meniscus tissue is developed; this can simplify the scaffold fabrication procedure and period. The transplantation of a porous meniscus scaffold combined with a postoperative regular exercise stimulus facilitated the regeneration of anisotropic meniscal fibrocartilaginous tissue and protected the joint cartilage from degeneration in an ovine subtotal meniscectomy model. Single-cell RNA sequencing and immunofluorescence co-staining analyses further confirmed the occurrence of EndMT during meniscal regeneration. EndMT-transformed cells gave rise to fibrochondrocytes, subsequently contributing to meniscal fibrocartilage regeneration. Thus, an efficient translational strategy to facilitate meniscal regeneration is developed.

Laminin Beta 2 Is Localized at the Sites of Blood-Brain Barrier and Its Disruption Is Associated With Increased Vascular Permeability, Histochemical, and Transcriptomic Study.

Heterotrimeric extracellular matrix proteins laminins are mostly deposited at basal membranes and are important in repair and neoplasia. Here, we localize laminin beta 2 (LAMB2) at the sites of blood-brain barrier (BBB). Microvasculature (MV) of normal brain is endowed with complete LAMB2 coverage. In contrast, its cognate protein laminin beta 1 (LAMB1) is absent in MV of normal brain but emerges at the sprouting tip of a growing vessels. Similarly, vascular proliferation in high-grade gliomas (HGG) is accompanied by marked overexpression of LAMB1, whereas LAMB2 shows deficient deposition. We find that many brain pathologies with presence of post-gadolinium enhancement (PGE) on magnetic resonance imaging (MRI) show disruption of LAMB2 vascular ensheathment. Inhibition of vascular endothelial growth factor signaling in HGG blocks angiogenesis, suppresses PGE in HGG, prevents expression of LAMB1, and restores LAMB2 vascular coverage. Analysis of single-cell RNA sequencing (scRNA-seq) databases shows that in quiescent brain LAMB2 is predominantly expressed by BBB-associated pericytes (PCs) and endothelial cells (ECs), whereas neither cell types produce LAMB1. In contrast, in HGG, both LAMB1 and 2 are overexpressed by endothelial precursor cells, a phenotypically unique immature group, specific to proliferating hyperplastic MV.

Targeted inhibition of CHKα and mTOR in models of pancreatic ductal adenocarcinoma: A novel regimen for metastasis

Pancreatic ductal adenocarcinoma (PDAC) is a highly metastatic malignancy for which there are currently no effective anti-metastatic therapies. Herein, we employed single-cell RNA sequencing and metabolomics analysis to demonstrate that metastatic cells highly express focal adhesion kinase (FAK), which promotes metastasis by remodeling choline kinase α (CHKα)-dependent choline metabolism. We designed a novel CHKα inhibitor, CHKI-03, and verified its efficacy in inhibiting metastasis in multiple preclinical models. Classical and newly synthesized small-molecule inhibitors have previously been used to assess the therapeutic potential of targeting mTOR and CHKα in various animal models. Mechanistically, FAK activated mTOR and its downstream HIF-1α, thereby elevating CHKα expression and promoting the proliferation, migration, and invasion of PDAC cells, as well as tumor growth and metastasis. Consistently, high expression levels of both FAK and CHKα are correlated with poor prognosis in patients with PDAC. Notably, CHK1-03 inhibited CHKα expression and also suppressed mTORC1 phosphorylation, disrupting the mTORC1-CHKα positive feedback loop. In addition, the combination of CHKI-03 and the mTORC1 inhibitor rapamycin synergistically inhibited tumor growth and metastasis in PDX models. The combination of CHKI-03 and rapamycin demonstrates considerable therapeutic efficacy in PDO models resistant to gemcitabine. Our findings reveal a pivotal mechanism underlying PDAC metastasis regulated by mTORC1-CHKα loop-dependent choline metabolism reprogramming, highlighting the therapeutic potential of this novel regimen for treating PDAC metastasis.

Single-cell RNA sequencing and immune repertoire analysis revealed dynamic immune characteristics associated with peripheral blood during sepsis

Sepsis is a potentially fatal condition arising from an abnormal immune response to an infection, which can result in organ failure and even death. To explore the mechanism underlying the dysregulated immune response during sepsis and identify potential therapeutic targets, single-cell RNA sequencing (scRNA-seq) and immune repertoire analysis were conducted to depict the cellular landscape of peripheral blood cells in septic mice. We observed significant alterations in the number and proportion of peripheral blood cell populations driven by sepsis. By combining single-cell gene expression profiles and B cell receptor (BCR) repertoire analysis, we discerned that infection inflicted serious damage on the antigen presentation ability of B cells and the diversity of BCR in a short time. In addition, we found that the cecal ligation and puncture procedure in mice inhibited the communication signals of CD4+ and CD8+ T cells and decreased the interactions between B cells and other cells. Our study provides detailed insights into the dynamic changes in the biological characteristics of peripheral blood cells driven by sepsis and provides important advances in our understanding of immune disorders during sepsis.

Validation of non-destructive morphology-based selection of cerebral cortical organoids by paired morphological and single-cell RNA-seq analyses.

Organoids, self-organized cell aggregates, contribute significantly to developing disease models and cell-based therapies. Organoid-to-organoid variations, however, are inevitable despite the use of the latest differentiation protocols. Here, we focused on the morphology of organoids formed in a cerebral organoid differentiation culture and assessed their cellular compositions by single-cell RNA sequencing analysis. The data revealed that organoids primarily composed of non-neuronal cells, such as those from the neural crest and choroid plexus, showed unique morphological features. Moreover, we demonstrate that non-destructive morphological analysis can accurately distinguish organoids composed of cerebral cortical tissues from other cerebral tissues, thus enhancing experimental accuracy and reliability to ensure the safety of cell-based therapies.

Alpha-2-macroglobulin mitigates glucocorticoid-induced osteonecrosis via Keap1/Nrf2 pathway activation

Glucocorticoids (GCs) are widely prescribed for various medical conditions, but prolonged use can result in osteonecrosis of the femoral head (ONFH), a serious condition characterized by bone tissue death due to reduced blood flow. Alpha-2-macroglobulin (A2M) is known to regulate oxidative stress and has been implicated in numerous biological processes. However, its role in GCs-induced ONFH has not been fully elucidated. This study investigates the involvement of A2M in ONFH by examining its activation of the Keap1/Nrf2 signaling pathway. Transcriptomic and proteomic analyses of patient samples with GCs-induced ONFH revealed a significant downregulation of A2M. A rat model of GCs-induced ONFH was then used to overexpress A2M, with subsequent evaluation through histopathological staining. Single-cell RNA sequencing and proteomic analysis indicated that A2M overexpression promotes the proliferation of anti-inflammatory macrophage clusters. Both in vivo and in vitro experiments demonstrated that A2M overexpression significantly alleviated ONFH symptoms by modulating oxidative stress and apoptosis via the Keap1/Nrf2 pathway. These findings underscore the critical role of A2M in mitigating GCs-induced ONFH, providing new therapeutic strategies and targets for future research.

Exploring the heterogeneity of interstitial cells of Cajal and their properties in gastrointestinal mesenchymal tumors applying single-cell RNA sequencing analysis

BackgroundGastrointestinal mesenchymal stromal tumors (GISTs) are a group of intramural tumors that exhibit a wide range of morphologies. Dysfunction or loss of interstitial cells of Cajal (ICCs) is correlated with the disorders of gastrointestinal motility. At present, the characterization and molecular mechanisms underlying the role of ICCs in GIST are still not clear.MethodsThe GSE162115 dataset from Gene Expression Omnibus database was processed using Seurat package for quality control, data normalization, and cell clustering. Differential expression and functional enrichment analyses were performed using the FindAllMarkers function and clusterProfiler package. Cellular heterogeneity was assessed by CytoTRACE and potential regulatory mechanisms of ICCs in GISTs were investigated using SCENIC. Cellular communication was inferred and analyzed applying the CellChat package.ResultsEight clusters were identified based on 34,861 cells. Intra-tumor samples had a higher proportion of ICCs than peri-tumor. ICCs were related to cell cycle and glycolytic activity in intra-tumor samples, while those in peri-tumor samples were involved in immune response. Further analysis identified four ICC subgroups (subcluster 1–4), of which subcluster 3 showed the most typical stem cell properties and interacted with the rest of the cells through the MIF-CD74 (CD44) protein.ConclusionThis study analyzed the heterogeneity and stem cell properties of ICCs in GISTs, revealing the molecular mechanisms and potential therapeutic targets for GISTs.

Metastasis of colon cancer requires Dickkopf-2 to generate cancer cells with Paneth cell properties.

Metastasis is the leading cause of cancer-related mortality. Paneth cells provide stem cell niche factors in homeostatic conditions, but the underlying mechanisms of cancer stem cell niche development are unclear. Here we report that Dickkopf-2 (DKK2) is essential for the generation of cancer cells with Paneth cell properties during colon cancer metastasis. Splenic injection of Dkk2 -knockout (KO) cancer organoids into C57BL/6 mice resulted in a significant reduction of liver metastases. Transcriptome analysis showed reduction of Paneth cell markers such as lysozymes in KO organoids. Single cell RNA sequencing analyses of murine metastasized colon cancer cells and patient samples identified the presence of lysozyme positive cells with Paneth cell properties including enhanced glycolysis. Further analyses of transcriptome and chromatin accessibility suggested Hepatocyte nuclear factor 4-alpha (HNF4A) as a downstream target of DKK2. Chromatin immunoprecipitation followed by sequencing analysis revealed that HNF4A binds to the promoter region of Sox9 , a well-known transcription factor for Paneth cell differentiation. In the liver metastatic foci, DKK2 knockout rescued HNF4A protein levels followed by reduction of lysozyme positive cancer cells. Taken together, DKK2-mediated reduction of HNF4A protein promotes the generation of lysozyme positive cancer cells with Paneth cell properties in the metastasized colon cancers.

HPV-driven heterogeneity in cervical cancer: study on the role of epithelial cells and myofibroblasts in the tumor progression based on single-cell RNA sequencing analysis.

Cervical cancer (CC) is a neoplasia with a high heterogeneity. We aimed to explore the characteristics of tumor microenvironment (TME) for CC treatment. HPV positive (+) and negative (-) samples from cervical cancer (CC) patients were sourced from the Gene Expression Omnibus (GEO) database. The single-cell RNA sequencing (scRNA-seq) data were processed and annotated for cell types utilizing the Seurat package. Following this, the expression levels and biological roles of the marker genes were analyzed applying real-time PCR (RT-PCR) and transwell assays. Furthermore, the enrichment of genes with significantly differential expressions and copy number variations was assessed by the ClusterProlifer and inferCNV software packages. Seven main cell clusters were classified based on a total of 12,431 cells. The HPV- CC samples exhibited a higher immune cell infiltration level, while epithelial cells and myofibroblasts had higher proportion in the HPV+ CC samples with extensive heterogeneity. Immune pathways including antigen treatment and presentation, immunoglobulin production and T cell mediated immunity were significantly activated in the HPV- CC group with lower cell cycle and proliferation activity. However, the anti-tumor immunity of these cells was inhibited in HPV+ CC group with higher cell proliferation activity. Moreover, the amplification and loss of CNVs also supported that these cells in HPV- CC samples were prone to anti-tumor activation. Further cell validation results showed that except GZMA, the levels of APOC1, CEACAM6, FOXP3, SFRP4 and TFF3 were all higher in CC cells Hela, and that silencing TFF3 could inhibit the migration and invasion of CC cells in-vitro. This study highlighted the critical role of HPV infection in CC progression, providing a novel molecular basis for optimizing the current preventive screening and personalized treatment for the cancer.

Pyroptosis-related genes features on prediction of the prognosis in liver cancer: An integrated analysis of bulk and single-cell RNA sequencing

ObjectiveThis study explores the impact of pyroptosis-related genes (PRG) on the prognosis of liver cancer (LC). Methods421 samples (371 tumor samples and 50 normal samples) from the Cancer Genome Atlas (TCGA) were included in this study. GSE14520 dataset (data of RNA expression and relevant clinicopathological features), GSE125449 dataset (single-cell data in LC) and HCCDB18 dataset (validation on the reliability of the model) were downloaded as appropriate. Download the PRG and its corresponding pathway information from the gene set enrichment analysis (GSEA) website. The consensus clustering was performed by ConsensusClusterPlus package. Differentially expressed genes (DEGs) were identified using limma package, and prognostic features were constructed using un/multivariate and Lasso Cox regression. Pathway enrichment analysis was conducted by ssGSEA method. Receiver Operating Characteristic and the survival analysis were conducted by timeROC and Survminer packages. The Seurat package was used for single-cell RNA sequencing (scRNA-seq) analysis. For cellular validation, following the quantification on the key genes via reverse-transcription quantitative PCR, the Transwell and scratch assays were applied to evaluate the in-vitro invasion and migration of LC cells Huh-7. Results12 prognosis-related genes were identified to be related to the progression of LC. Three subtypes including C1, C2 and C3 were categorized using the 12 prognosis-related genes and PRGs significantly related to the prognosis of LC patients. The worst and best prognosis was seen in C3 subtype and C2 subtype, respectively. Hallmark pathway enrichment analysis has shown the concurrent immunoactivation and immune escape in C3 subtype. A RiskScore model was constructed using 8 key genes (KPNA2, UCK2, FTCD, CBX2, RAB32, HMMR, S100A9 and ANXA10) from the DEGs of three subtypes. The RiskScore system as an independent prognostic factor dividing the patients into high and low risk groups, and patients of the high-risk group had poor prognosis in both test set and validation set. A nomogram model combining the risk score had the extreme higher benefit. Further, 6 subclusters were identified from scRNA-seq analysis, where the highest PYROPTOSIS score was seen in Monocytic-Macrophages. The quantification on the key genes has suggested the high expressions of KPNA2, UCK2, CBX2, RAB32, HMMR and S100A9 and the low expressions of FTCD and ANXA10 in LC cells Huh-7. Particularly, UCK2 knockdown evidently diminished the number of invaded and migrated LC cells in vitro. ConclusionThe risk model associated with pyproptosis is crucial for the tumor immunity of LC and may serve as a prognostic indicator for patients suffering from LC. Our findings will offer new perspectives for immunotherapies targeting LC.

MIR4435-2HG: A novel biomarker for triple-negative breast cancer diagnosis and prognosis, activating cancer-associated fibroblasts and driving tumor invasion through EMT associated with JNK/c-Jun and p38 MAPK signaling pathway activation

BackgroundBreast cancer has the highest incidence rate and causes the most fatalities among all female cancers worldwide. Triple-negative breast cancer (TNBC) is known for its strong invasiveness and higher rates of recurrence. In this research, we aimed to identify MIR4435-2HG as a promising long non-coding RNA (lncRNA) biomarker and therapeutic target for TNBC. MethodsUtilizing clinicopathological information and transcriptome data from The Cancer Genome Atlas (TCGA) database, we assessed the clinical relevance of MIR4435-2HG in breast cancer through univariate and multivariate COX regression, receiver operating characteristic (ROC) analysis, as well as Kaplan-Meier survival analysis. To investigate the biological role of MIR4435-2HG in TNBC, we conducted gene set enrichment analysis (GSEA), as well as Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses. Additionally, we constructed and validated a nomogram to predict disease-free survival (DFS). Both the R package “pRRophetic” and the Tumor Immune Dysfunction and Exclusion (TIDE) algorithm were employed to forecast the sensitivity to different therapeutics between the high- and low-MIR4435-2HG groups. We employed single-cell RNA sequencing analysis and tumor microenvironment infiltration analysis to investigate the potential involvement of MIR4435-2HG in the TNBC tumor microenvironment. Cellular biological behaviors were assessed utilizing CCK-8, transwell assays, and wound-healing assays. Furthermore, we performed RNA-seq, qRT-PCR, and western blotting analyses to elucidate and confirm the specific mechanisms underlying the role of MIR4435-2HG in TNBC. ResultsIn our study, we have identified MIR4435-2HG as a significant diagnostic and prognostic factor for TNBC. We observed that MIR4435-2HG is widely expressed and might have a significant impact on the reshaping of the TNBC tumor microenvironment. Patients with TNBC in the high-MIR4435-2HG group may show reduced sensitivity to cisplatin, doxorubicin, and gemcitabine and have an increased propensity for immune escape. Knockdown of MIR4435-2HG inhibits cancer-associated fibroblasts (CAFs) activation. Notably, MIR4435-2HG predominantly enhances the migratory and invasive capabilities of TNBC cells through the epithelial-mesenchymal transition (EMT) process. Mechanistically, we validated that MIR4435-2HG activates the JNK/c-Jun and p38 non-classical MAPK signaling pathway in TNBC cells. ConclusionsOur findings highlight the significant potential of MIR4435-2HG as a highly promising biomarker for TNBC. Targeting MIR4435-2HG could represent an appealing therapeutic approach for TNBC.

Abstract C105: Novel cancer-specific hypoxia signature predicts poor prognosis and highlights racial disparities in breast cancer

Abstract Triple-negative breast cancer (TNBC) is the most aggressive subtype of breast cancer. TNBC displays exceptional transcriptional heterogeneity, both between patients and intratumorally, and disproportionately affects underserved women. Hypoxia, a state of low oxygen levels, is often present in the solid tumor microenvironment. In TNBC, hypoxia is a key driver of aggressiveness and therapeutic resistance. Past bulk RNA sequencing (RNAseq) studies in cancer research have lacked spatial resolution, failing to uncover precise transcriptional differences within tumor regions. This spatial limitation has been especially problematic for studying transcriptional heterogeneity of the cellular hypoxia response within tumors. By integrating single-cell RNA sequencing and spatial transcriptomic analyses in a TNBC xenograft tumor model, our research has uncovered a novel hypoxia gene signature, specific to cancer cells. Elevated expression of this hypoxia signature predicts poorer prognosis in breast cancer patients, especially within TNBC patients and those of African ancestry. This suggests that hypoxia may play a role in the racial disparities observed in TNBC prognosis, potentially informing treatment strategies. This workflow introduces a novel framework for translating general spatial transcriptomics data into clinically relevant and accurate gene signatures. Citation Format: Kent Schechter, Long C. Nguyen, Geetha P. Yerradoddi, Rania Bassiouni, John D. Carpten, Marsha R. Rosner. Novel cancer-specific hypoxia signature predicts poor prognosis and highlights racial disparities in breast cancer [abstract]. In: Proceedings of the 17th AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2024 Sep 21-24; Los Angeles, CA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2024;33(9 Suppl):Abstract nr C105.

YTHDF1 in periaqueductal gray inhibitory neurons contributes to morphine withdrawal responses in mice

BackgroundPhysical symptoms and aversion induced by opioid withdrawal strongly affect the management of opioid addiction. YTH N6-methyladenosine (m6A) RNA binding protein 1 (YTHDF1), an m6A-binding protein, from the periaqueductal gray (PAG) reportedly contributes to morphine tolerance and hyperalgesia. However, the role of YTHDF1 in morphine withdrawal remains unclear.MethodsA naloxone-precipitated morphine withdrawal model was established in C57/BL6 mice or transgenic mice. YTHDF1 was knocked down via adeno-associated virus transfection. Combined with the results of the single-cell RNA sequencing analysis, the changes in morphine withdrawal somatic signs and conditioned place aversion (CPA) scores were compared when YTHDF1 originating from different neurons in the ventrolateral periaqueductal gray (vlPAG) was knocked down. We further explored the role of inflammatory factors and transcription factors related to inflammatory response in morphine withdrawal.ResultsOur results revealed that YTHDF1 expression was upregulated in the vlPAG of mice with morphine withdrawal and that the knockdown of vlPAG YTHDF1 attenuated morphine withdrawal-related somatic signs and aversion. The levels of NF-κB and p-NF-κB were reduced after the inhibition of YTHDF1 in the vlPAG. YTHDF1 from vlPAG inhibitory neurons, rather than excitatory neurons, facilitated morphine withdrawal responses. The inhibition of YTHDF1 in vlPAG somatostatin (Sst)-expressing neurons relieved somatic signs of morphine withdrawal and aversion, whereas the knockdown of YTHDF1 in cholecystokinin (Cck)-expressing or parvalbumin (PV)-expressing neurons did not change morphine withdrawal-induced responses. The activity of c-fos + neurons, the intensity of the calcium signal, the density of dendritic spines, and the frequency of mIPSCs in the vlPAG, which were increased in mice with morphine withdrawal, were decreased with the inhibition of YTHDF1 from vlPAG inhibitory neurons or Sst-expressing neurons. Knockdown of NF-κB in Sst-expressing neurons also alleviated morphine withdrawal-induced responses.ConclusionsYTHDF1 originating from Sst-expressing neurons in the vlPAG is crucial for the modulation of morphine withdrawal responses, and the underlying mechanism might be related to the regulation of the expression and phosphorylation of NF-κB.

Immune and inflammatory insights in atherosclerosis: development of a risk prediction model through single-cell and bulk transcriptomic analyses.

Investigation into the immune heterogeneity linked with atherosclerosis remains understudied. This knowledge gap hinders the creation of a robust theoretical framework essential for devising personalized immunotherapies aimed at combating this disease. Single-cell RNA sequencing (scRNA-seq) analysis was employed to delineate the immune cell-type landscape within atherosclerotic plaques, followed by assessments of cell-cell interactions and phenotype characteristics using scRNA-seq datasets. Subsequently, pseudotime trajectory analysis was utilized to elucidate the heterogeneity in cell fate and differentiation among macrophages. Through integrated approaches, including single-cell sequencing, Weighted Gene Co-expression Network Analysis (WGCNA), and machine learning techniques, we identified hallmark genes. A risk score model and a corresponding nomogram were developed and validated using these genes, confirmed through Receiver Operating Characteristic (ROC) curve analysis. Additionally, enrichment and immune characteristic analyses were conducted based on the risk score model. The model's applicability was further corroborated by in vitro and in vivo validation of specific genes implicated in atherosclerosis. This comprehensive scRNA-seq analysis has shed new light on the intricate immune landscape and the role of macrophages in atherosclerotic plaques. The presence of diverse immune cell populations, with a particularly enriched macrophage population, was highlighted by the results. Macrophage heterogeneity was intricately characterized, revealing four distinct subtypes with varying functional attributes that underscore their complex roles in atherosclerotic pathology. Intercellular communication analysis revealed robust macrophage interactions with multiple cell types and detailed pathways differing between proximal adjacent and atherosclerotic core groups. Furthermore, pseudotime trajectories charted the developmental course of macrophage subpopulations, offering insights into their differentiation fates within the plaque microenvironment. The use of machine learning identified potential diagnostic markers, culminating in the identification of RNASE1 and CD14. The risk score model based on these biomarkers exhibited high accuracy in diagnosing atherosclerosis. Immune characteristic analysis validated the risk score model's efficacy in defining patient profiles, distinguishing high-risk individuals with pronounced immune cell activities. Finally, experimental validation affirmed RNASE1's involvement in atherosclerotic progression, suggesting its potential as a therapeutic target. Our findings have advanced our understanding of atherosclerosis immunopathology and paved the way for novel diagnostic and therapeutic strategies.

In vivo CRISPR screens identify Mga as an immunotherapy target in triple-negative breast cancer

Immune evasion is not only critical for tumor initiation and progression, but also determines the efficacy of immunotherapies. Through iterative in vivo CRISPR screens with seven syngeneic tumor models, we identified core and context-dependent immune evasion pathways across cancer types. This valuable high-confidence dataset is available for the further understanding of tumor intrinsic immunomodulators, which may lead to the discovery of effective anticancer therapeutic targets. With a focus on triple-negative breast cancer (TNBC), we found that Mga knock-out significantly enhances antitumor immunity and inhibits tumor growth. Transcriptomics and single-cell RNA sequencing analyses revealed that Mga influences various immune-related pathways in the tumor microenvironment. Our findings suggest that Mga may play a role in modulating the tumor immune landscape, though the precise mechanisms require further investigation. Interestingly, we observed that low MGA expression in breast cancer patients correlates with a favorable prognosis, particularly in those with active interferon-γ signaling. These observations provide insights into tumor immune escape mechanisms and suggest that further exploration of MGA's function could potentially lead to effective therapeutic strategies in TNBC.

Revealing the association between East Asian oral microbiome and colorectal cancer through Mendelian randomization and multi-omics analysis.

Colorectal cancer (CRC) poses a global health threat, with the oral microbiome increasingly implicated in its pathogenesis. This study leverages Mendelian Randomization (MR) to explore causal links between oral microbiota and CRC using data from the China National GeneBank and Biobank Japan. By integrating multi-omics approaches, we aim to uncover mechanisms by which the microbiome influences cellular metabolism and cancer development. We analyzed microbiome profiles from 2017 tongue and 1915 saliva samples, and GWAS data for 6692 CRC cases and 27178 controls. Significant bacterial taxa were identified via MR analysis. Single-cell RNA sequencing and enrichment analyses elucidated underlying pathways, and drug predictions identified potential therapeutics. MR identified 19 bacterial taxa significantly associated with CRC. Protective effects were observed in taxa like RUG343 and Streptococcus_umgs_2425, while HOT-345_umgs_976 and W5053_sp000467935_mgs_712 increased CRC risk. Single-cell RNA sequencing revealed key pathways, including JAK-STAT signaling and tyrosine metabolism. Drug prediction highlighted potential therapeutics like Menadione Sodium Bisulfite and Raloxifene. This study establishes the critical role of the oral microbiome in colorectal cancer development, identifying specific microbial taxa linked to CRC risk. Single-cell RNA sequencing and drug prediction analyses further elucidate key pathways and potential therapeutics, providing novel insights and personalized treatment strategies for CRC.

Berberine Targets PKM2 to Activate the t-PA-Induced Fibrinolytic System and Improves Thrombosis.

Arterial thrombosis, a condition in which thrombi form in arteries, can lead to various acute cardiovascular diseases and impact the quality of life and survival of patients. Berberine (BBR), a quaternary ammonium alkaloid, has been shown to treat these diseases. However, further exploration is needed to understand underlying mechanisms of BBR. Rats were administered BBR via intramuscular injection. Then, an FeCl3-coated filter paper was applied to a carotid artery to induce thrombosis. The size of the thrombus and the blood flow velocity were evaluated by carotid ultrasound. The shape of the thrombus was observed using staining and microscopy. The expression levels of mRNA and proteins were verified. Additionally, mass spectrometry and single-cell RNA sequencing analysis were conducted. The administration of BBR resulted in a significant reduction in the thrombus area and an extension of the thrombus-clogging time. Furthermore, BBR administration effectively reversed the decreasing tissue-plasminogen activator (t-PA) expression and alterations in fibrinolysis system of model group. Additionally, the expression of PKM2 was suppressed following BBR administration, and the overexpression of PKM2 inhibited t-PA expression. BBR ameliorates thrombosis by modulating expression of PKM2, subsequently impacting the expression of t-PA within fibrinolytic system. These preliminary findings suggest that BBR could be a potential preventive and therapeutic strategy for arterial thromboembolic diseases.