Cell Subtypes Research Articles

Prediction of cardiovascular outcomes by subtle changes in the peripheral immune cell landscape - insights from the LURIC single cell RNA-sequencing study (LuRNA)

Abstract Background and Aims Atherosclerosis and its clinical sequelae, myocardial infarction and stroke, represent the main causes of death worldwide. Although preclinical evidence has suggested the existence of an inflammatory response driving disease, the extend of cellular alterations in human atherosclerosis remains enigmatic. Here, we employ single cell RNA-sequencing (scRNA-seq) on peripheral blood mononucleated cells (PBMCs) in a well-defined cardiovascular risk cohort from the "Ludwigshafen Risk and Cardiovascular Health (LURIC) trial" to define changes in the immune cell landscape in atherosclerotic patients. Methods and Results Of 3317 patients enrolled in the LURIC trial between 1997 and 2000, we selected individuals with stable coronary-artery disease (CAD) (n=31) and healthy patients (no CAD) (n=16) by propensity score matching, adjusted for CRP, NTproBNP, Troponin T, LDL-C, and Cystatin-C, in a case-control design. scRNA-seq was performed by droplet sequencing on PBMCs stored in liquid nitrogen. Individual data sets were integrated and changes between healthy individuals and patients with CAD were evaluated on numeric and transcriptional level. Single cell transcriptomes were annotated using an established CITE-seq reference atlas. While bulk RNA-sequencing of PBMC preparations revealed only minor changes between both conditions, we identified several leukocyte populations on a single cell level with specific transcriptomes that were differentially regulated between both conditions. Interestingly, we found that several subsets of Natural Killer (NK) cells, T cells with an enrichment of proliferation-associated pathways, and a population resembling hematopoietic stem cells were more frequent in patients with CAD, while naïve phenotypes of B and T cells were overrepresented in the absence of CAD. Consistently, most T and B cell populations in patients with CAD were enriched in pathways associated with cell activation, immune receptor signalling, and differentiation. In addition, we detected restricted repertoires of T cell receptor sequences in several adaptive immune cell subtypes from patients with CAD, suggestive of increased cellular clonality and antigen-specificity. Finally, in a comparison of cellular frequencies with cardiovascular outcomes over more than 15 years, several immune cell subsets and cell-type specific transcriptional programs predicted cardiovascular death in this case-control scenario in multi-variate adjusted regression analysis. Notably, addition of scRNA-seq derived parameters was superior to traditional risk prediction with clinical characteristics and soluble biomarkers alone. Conclusion Employing scRNAseq, we demonstrate that atherosclerosis is associated with a profound change in the circulating immune cell landscape even in the absence of measurable differences in inflammatory biomarkers. These findings propose the usage of scRNAseq for the discovery of outcome-relevant cellular biomarkers in the future.

Identification of a Novel Signature Based on Ferritinophagy-Related Genes to Predict Prognosis in Lung Adenocarcinoma: Focus on AHNAK2

Background: Lung adenocarcinoma (LUAD) accounts for over 40% of all non-small cell lung cancer (NSCLC) cases and continues to be difficult to treat despite advancements in diagnostics and therapies. Ferritinophagy, a newly recognized autophagy process linked to ferroptosis, has been associated with LUAD development. Recent studies have shown a dysregulation of genes related to ferritinophagy in LUAD, indicating its potential as a therapeutic target. Methods: We constructed a predictive model using seven genes associated with ferritinophagy. The model’s accuracy was evaluated across three independent gene expression datasets. We analyzed the biological functions, immune environment, mutations, and drug sensitivities in groups with high and low risk. Utilizing a single-cell sequencing (scRNA-seq) dataset, we confirmed the expression of the model genes and identified a subtype of epithelial cells expressing AHNAK2. We further investigated the impact of the ferritinophagy-related gene AHNAK2 on LUAD cell proliferation, invasion, migration, and ferroptosis in vitro. Results: Our prediction model, comprising seven genes (AHNAK2, ARNTL2, CD27, LTB, SLC15A1, SLC2A1, and SYT1), has shown potential in predicting the prognosis of individuals diagnosed with LUAD. Notably, AHNAK2 impedes ferroptosis, promoting LUAD progression in vitro. Conclusions: Our research suggests that ferritinophagy-associated genes are promising prognostic markers for LUAD and lay the groundwork for further exploration of ferritinophagy’s role in LUAD. Furthermore, we present AHNAK2 as a novel regulator of ferroptosis, which requires further investigation to understand its mechanism.

Measurement of Four Uterine NK Cell Subtypes Using Multiplexed Fluorescent Immunohistochemical Staining in Women with Repeated Implantation Failure

Measurement of Four Uterine NK Cell Subtypes Using Multiplexed Fluorescent Immunohistochemical Staining in Women with Repeated Implantation Failure

Nanoplastics: Immune Impact, Detection, and Internalization after Human Blood Exposure by Single-Cell Mass Cytometry.

The increasing exposure to nanoplastics (NPs) raises significant concerns for human health, primarily due to their potential bioaccumulative properties. While NPs have recently been detected in human blood, their interactions with specific immune cell subtypes and their impact on immune regulation remain unclear. In this proof-of-concept study, model palladium-doped polystyrene NPs (PS-Pd NPs) are utilized to enable single-cell mass cytometry (CyTOF) detection. The size-dependent impact of carboxylate polystyrene NPs (50-200 nm) is investigated across 15 primary immune cell subpopulations using CyTOF. By taking advantage of Pd-doping for detecting PS-Pd NPs, this work evaluates their impact on human immune-cells at the single-cell level following blood exposure. This work traces PS-Pd NPs in 37 primary immune-cell subpopulations from human blood, quantifying the palladium atom count per cell by CyTOF while simultaneously assessing the impact of PS-Pd NPs on cell viability, functionality, and uptake. These results demonstrate that NPs can interact with, interfere with, and translocate into several immune cell subpopulations after exposure. In vivo distribution experiments in mice further confirmed their accumulation in immune cells within the liver, blood, and spleen, particularly in monocytes, macrophages, and dendritic cells. These findings provide valuable insights into the impact of NPs on human health.

How Co-Stimulatory/Inhibitory Molecules Vary Across Immune Cell Subtypes in the Severity of Systemic Lupus Erythematosus Compared to Controls

Background: Co-stimulatory and co-inhibitory molecules are critical to T cell responses and involved in the pathogenesis of systemic lupus erythematosus (SLE). This study aimed to comprehensively analyze the surface expression of these molecules in various phenotypic immune cells, comparing the differences between various levels of the severity in SLE and control groups. Methods: Peripheral blood mononuclear cells (PBMCs) were isolated using Ficoll-Paque from blood samples of severe SLE patients (treatment with immunosuppressants), mild SLE patients (excluding those with persistent proteinuria or thrombocytopenia), and healthy controls (n = 10 each). PBMCs were stimulated for 48 h. The cells were stained with anti-CD3, CD4, CD28, PD-1, and CTLA-4 antibodies and analyzed by flow cytometry. Differences between groups were assessed using the Kruskal–Wallis test and Mann–Whitney U-test, with median values and statistical significance (p < 0.05) reported. Results: The results showed that CD28 expression was significantly higher in SLE patients compared to controls, with the highest levels in mild SLE. However, CD3+ CD28+ and CD4+ CD28+ cells were more prevalent in controls (p = 0.032 and 0.017, respectively). Mild SLE patients exhibited the highest CTLA-4 expression, with significant differences from severe SLE and controls (p = 0.030 and 0.037, respectively). PD-1 expression was lowest in severe SLE but highest in mild SLE within CD3+ CD4+ cells (p = 0.001). After 48 h of activation, CD4+ CTLA4+ and CD3+ CTLA4+ expression levels were significantly higher in controls compared to SLE groups. Conclusions: Our study highlighted that the expression of CD28, CTLA-4, and PD-1 in lymphocytes and specific T cell subsets was various according the severity of SLE in patients, underscoring their roles in disease pathogenesis.

Primary central nervous system lymphoma: a series report and literature review

ObjectiveThis study aims to analyze the clinical features, Radiological findings and diagnostic characteristics (tissue biopsy, flow cytometry and cerebrospinal fluid cytology) of primary central nervous system lymphoma (PCNSL). To improve the diagnosis of the disease.MethodsA total of 19 patients with primary central nervous system lymphoma admitted to the Second Hospital of Hebei Medical University from 2014 to 2023 were selected. The clinical data of the patients were retrospectively analyzed. Combined with the relevant literature, the clinical characteristics of PCNSL patients were analyzed to assist the diagnosis.ResultMost of the patients were middle-aged and elderly patients (65 years old), and the main clinical manifestations showed no obvious specificity, including headache, dizziness and limb weakness. Radiological findings showed that common site of currence were located in the brain parenchyma. The PCNSL subtype of 19 patients was all diffuse large B cell subtype by cytology, flow cytometry or histopathology. Six cases were diagnosed by biopsy pathology and 11 cases were diagnosed by flow cytometry of cerebrospinal fluid. In all patients diagnosed with PCNSL by clinical cytology (CSF cytology and flow cytometry), abnormal cells were found on CSF examination.ConclusionThe pathological type of primary central nervous system lymphoma was diffuse large B-cell subtype, with diverse clinical manifestations and multiple. Both tissue directed biopsy and flow cytometry could effectively assist clinical diagnosis. Early and timely diagnosis and intervention are of great significance for delaying the disease process and reducing the economic burden of patients and families.

Black pepper extract reduces tumour incidence via suppressing tumour-promoted cytokines/chemokines and modulating T cell subtype

ABSTRACT Our study aimed to evaluate tumour retardation effects of low-piperine fractional Piper nigrum extract (PFPE or LP-PE) related to enhancement of immune response. Results showed that PFPE at doses of 50 (low), 100 (intermediated) and 150 (high) mg/kg BW had a tumour incidence of 20, 20 and 0%, respectively. PFPE at low dose inhibited cytokines/chemokines in the early stages of cancer however it could not suppress longer than 3 months. Intermediated dose of PFPE inhibited cancer cytokines/chemokines including sICAM-1, CX3CL1, CXCL7, VEGF, TIMP-1, CCL5, CCL20, CXCL1, CXCL5, CXCL9 and IL-1β. Interestingly, PFPE stimulated IFN- γ at 3 months after treatment in all doses. Intermediated and high doses of PFPE increased Th1 and reduced Th2. Moreover, only a high dose of PFPE reduced Treg. Additionally, PFPE reduced ER-α and NF-κB and increased IL-6R and STAT3 proteins in tumour tissues. Our findings indicate that PFPE suppressed breast cancer via immune modulation.

Retinal Ganglion Cell Subtypes and Their Vulnerability in Glaucoma.

The detection of selective retinal ganglion cell damage in glaucoma has been a long sought-after goal, not just for the development of clinical tests for the early detection of glaucoma but for the elucidation of potential mechanisms underlying retinal ganglion cell loss. Early reports of the selective vulnerability of larger retinal ganglion cells (RGCs) in human studies did not translate simply to the loss of a particular class of RGC but more likely reflected shrinkage and degeneration across all RGC classes. Subsequent studies of nonhuman primate (NHP) models of glaucoma indicated some selectivity with great damage to the magnocellular vs parvocellular pathways. More recently, rodent models of experimental glaucoma have highlighted a selective vulnerability of OFF-centered RGCs-particularly those with transient responses. Selectivity for OFF pathway damage is also seen as a trend in a rat model of glaucoma. These data support the concept that some RGCs are more vulnerable to the effects of glaucoma damage. This chapter covers some of the methods to elucidate RGC damage and the relevance of model selection to mimic human glaucoma rather than just RGC death.

Exploring the Blood Biomarkers and Potential Therapeutic Agents for Human Acute Mountain Sickness Based on Transcriptomic Analysis, Inflammatory Infiltrates and Molecular Docking.

A high-altitude, low-pressure hypoxic environment has severe effects on the health and work efficiency of its residents, and inadequate preventive measures and adaptive training may lead to the occurrence of AMS. Acute exposure to hypoxia conditions can have a less-favorable physiological effect on the human immune system. However, the regulation of the immune system in high-altitude environments is extremely complex and remains elusive. This study integrated system bioinformatics methods to screen for changes in immune cell subtypes and their associated targets. It also sought potential therapeutically effective natural compound candidates. The present study observed that monocytes, M1 macrophages and NK cells play a crucial role in the inflammatory response in AMS. IL15RA, CD5, TNFSF13B, IL21R, JAK2 and CXCR3 were identified as hub genes, and JAK2 was positively correlated with monocytes; TNFSF13B was positively correlated with NK cells. The natural compound monomers of jasminoidin and isoliquiritigenin exhibited good binding affinity with JAK2, while dicumarol and artemotil exhibited good binding affinity with TNFSF13B, and all are expected to become a potential therapeutic agents.

Myeloid Mir34a suppresses colitis-associated colon cancer: characterization of mediators by single-cell RNA sequencing.

We have previously shown that general deletion of the gene encoding the p53-inducible Mir34a microRNA enhances the number and invasion of colitis-associated colorectal cancers (CACs) in mice. Since the p53-pathway has been implicated in tumor-suppression mediated by cells in the tumor microenvironment (TME) we deleted Mir34a in myeloid cells and characterized CACs in these with scRNA-Seq (single cell RNA sequencing). This revealed an increase inspecificmacrophage subtypes, such as Cdk8+ macrophagesand Mrc1+,M2-like macrophages. The latterdisplayed elevated expression of 21 known Mir34a target mRNAs, including Csf1r, Axl, Foxp1, Ccr1, Nampt, and Tgfbr2, and 32 predicted Mir34a target mRNAs. Furthermore, Mir34a-deficient BMDMs showed enhanced migration, elevated expression of Csf1r and a shift towards M2-like polarization when compared to Mir34a-proficient BMDMs. Concomitant deletion of Csf1r or treatment with a Csf1r inhibitor reduced the CAC burden and invasion in these mice. Notably, loss of myeloid Mir34a function resulted in a prominent, inflammatory CAC cell subtype, which displayed epithelial and macrophage markers. These cells displayed high levels of the EMT transcription factor Zeb2 and may therefore enhance the invasiveness of CACs. Taken together, our results provide in vivo evidence for a tumor suppressive role of myeloid Mir34a in CACs which is, at least in part, mediated by maintaining macrophages in an M1-like state via repression of Mir34a targets, such as Csf1r. Collectively, these findings may serve to identify new therapeutic targets and approaches for treatment of CAC.

LMD: Cluster-Independent Multiscale Marker Identification in Single-cell RNA-seq Data.

Identifying accurate cell markers in single-cell RNA-seq data is crucial for understanding cellular diversity and function. Localized Marker Detector (LMD) is a novel tool to identify "localized genes" - genes exclusively expressed in groups of highly similar cells - thereby characterizing cellular diversity in a multi-resolution and fine-grained manner. LMD constructs a cell-cell affinity graph, diffuses the gene expression value across the cell graph, and assigns a score to each gene based on its diffusion dynamics. LMD's candidate markers can be grouped into functional gene modules, which accurately reflect cell types, subtypes, and other sources of variation such as cell cycle status. We apply LMD to mouse bone marrow and hair follicle dermal condensate datasets, where LMD facilitates cross-sample comparisons, identifying shared and sample-specific gene signatures and novel cell populations without requiring batch effect correction or integration methods. Furthermore, we assessed the performance of LMD across nine single-cell RNA sequencing datasets, compared it with six other methods aimed at achieving similar objectives, and found that LMD outperforms the other methods evaluated.

P10.09.A BLOOD-BASED CLASSIFICATION OF IDH-STATUS OF NON-ENHANCING GLIOMAS: A MACHINE LEARNING APPROACH

Abstract BACKGROUND Non-invasive determination of IDH mutational status in patients with glioma could offer significant therapeutic opportunities. While IDH wildtype tumors (e.g., GBM) typically show enhancement on MRI, IDH mutant tumors often lack this enhancement. However, relying solely on anatomic radiology may lead to misclassification, and currently, tissue acquisition is the primary method for assessing IDH status in gliomas. This limitation hampers the development of neoadjuvant or intraoperative therapeutic strategies based on IDH status; thus, more minimally invasive methods to determine IDH status are needed. In this study, we assessed immune cell proportions, which vary by glioma subtype, from pre-surgery peripheral blood samples as an alternative method of classifying the IDH status in gliomas without enhancement. MATERIAL AND METHODS First, we employed a highly accurate large language model (GPT-4-Turbo-128k) with over 99% accuracy to read radiology notes and exclude patients with enhancing gliomas. Then, we identified twelve immune cell subtypes from whole blood using deconvolution algorithms based on DNA methylation data. These immune cell subtypes, along with patient age, were integrated into a machine learning model (random forest) to predict IDH status (wildtype vs. mutant), leveraging conditional Generative Adversarial Networks to generate synthetic data and mitigate bias in the datasets. Two independent datasets were included for training and validating the model. RESULTS The random forest model had an area under the ROC curve (AUC) of 0.90 and accurately identified IDH status in 81% of a training set of 287 gliomas (65 wildtype and 222 mutant tumors at varied time points after diagnosis). In an independent validation data set of 99 gliomas (6 wildtype and 93 mutant) from pre-surgery blood samples, the AUC was 0.93, and accuracy predicted IDH status in 92% of cases with a sensitivity of 93% and specificity of 83%. CONCLUSION We could accurately predict IDH status in pre-surgical peripheral blood samples from patients with non-enhancing gliomas. This minimally invasive approach is a promising step toward reducing risk and exploring opportunities for earlier therapies based on IDH status.

Lung Immune Cell Niches and the Discovery of New Cell Subtypes.

Immune cells in the lungs are important for maintaining lung function. The importance of immune cells in defending against lung diseases and infections is increasingly recognized. However, a primary knowledge gaps in current studies of lung immune cells is the understanding of their subtypes and functional heterogeneity. Increasing evidence supports the existence of novel immune cell subtypes that engage in the complex crosstalk between lung-resident immune cells, recruited immune cells, and epithelial cells. Therefore, further studies on how immune cells respond to perturbations in the pulmonary microenvironment are warranted. This review explores the processes behind the formation of the immune cell niche during lung development, and the characteristics and cell interaction modes of several major lung-resident immune cells. It indicates that distinct lung microenvironments or inflammatory niches can mediate the formation of different cell subtypes. These findings summarize and clarify paths to identify new cell subtypes that originate from resident progenitor cells and recruited peripheral cells, which are remodeled by the pulmonary microenvironment. The development of new techniques combining transcriptome analysis and location information is essential for identifying new immune cell subtypes and their relative immune niches, as well as for uncovering the molecular mechanisms of immune cell-mediated lung homeostasis.

Cytokine-armed vaccinia virus promotes cytotoxicity toward pancreatic carcinoma cells via activation of human intermediary CD56dimCD16dim natural killer cells.

Pancreatic ductal adenocarcinoma (PDAC) remains a particularly aggressive disease with few effective treatments. The PDAC tumor immune microenvironment (TIME) is known to be immune suppressive. Oncolytic viruses can increase tumor immunogenicity via immunogenic cell death (ICD). We focused on tumor-selective (vvDD) and cytokine-armed Western-reserve vaccinia viruses (vvDD-IL2 and vvDD-IL15) and infected carcinoma cell lines as well as patient-derived primary PDAC cells. In co-culture experiments, we investigated the cytotoxic response and the activation of human natural killer (NK). Infection and virus replication were assessed by measuring virus encoded YFP. We then analyzed intracellular signaling processes and oncolysis via in-depth proteomic analysis, immunoblotting and TUNEL assay. Following the co-culture of mock or virus infected carcinoma cell lines with allogenic PBMCs or NK cell lines, CD56+ NK cells were analyzed with respect to their activation, cytotoxicity and effector function. Both, dose- and time-dependent release of danger signals following infection weremeasured. Viruses effectively entered PDAC cells, emitted YFP signals and resulted in concomitant oncolysis. The proteome showed reprogramming of normally active core signaling pathways in PDAC (e.g., MAPK-ERK signaling). Danger-associated molecular patterns were released upon infection and stimulated co-cultured NK cells for enhanced effector cytotoxicity. NK cell subtyping revealed enhanced numbers and activation of a rare CD56dimCD16dim population. Tumor cell killing was primarily triggered via Fas ligands rather than granule release, resulting in marked apoptosis. Overall, the cytokine-armed vaccinia viruses induced NK cell activation and enhanced cytotoxicity toward human PDAC cells in vitro. We could show that cytokine-armed virus targets the carcinoma cells and thus hasgreat potential to modulate the TIME in PDAC.

Rhoifolin Attenuates Concanavalin A-Induced Autoimmune Hepatitis in Mice via JAKs/STATs Mediated Immune and Apoptotic Processes.

Rhoifolin (ROF) exhibits a diverse range of biological activities, encompassing anticancer, hepatoprotective, antidiabetic, antirheumatic, and antiviral properties. However, the specific protective effects and possible mechanisms of the compound against T-cell-mediated autoimmune hepatitis have not been previously elucidated. In the present study, adult male mice were administered Con A (20 mg/kg, intravenously) for 8 h. In the treated groups, mice were pretreated with ROF daily (20 mg/kg and 40 mg/kg, orally) for 7 days before Con A intoxication. The results showed that ROF significantly decreased serum biochemical indices (ALT, AST, ALP, and LDH) and regulated related oxidative stress indicators (MDA, SOD, and GSH), reduced hepatic necrosis areas and immune cells infiltration, inhibited the release of various inflammatory factors (TNF-α, IFN-γ, IL-2, and IL-17), and improved hepatic tissue apoptosis, thereby alleviating hepatic damage induced by Con A. Additionally, we have also confirmed that ROF efficiently inhibited Th1/Th17 cells polarization via modulation of the JAK2/JAK3/STAT1/STAT3 signaling pathways both in vivo and in vitro. Moreover, the molecular mechanism examination also demonstrated that ROF regulated apoptotic cascade signaling through IL-6/JAK2/STAT1/STAT3 controlling BNIP3 activity in primary hepatocytes. These effects were in good agreement with the bioinformatics analysis of ROF treatment for AIH. In conclusion, our findings provide new insights into the potential use of ROF for AIH therapy, which may result from the specific regulation of the T cell subtype polarization and the apoptosis of liver cells via modulation of the JAKs/STATs signaling pathways.

Single-Cell Transcriptomics Reveals Cellular Heterogeneity and Drivers in Serrated Pathway-Driven Colorectal Cancer Progression

Serrated lesions are common precancerous pathways in colorectal cancer (CRC), but the process by which they progress to malignancy remains unclear. We aimed to elucidate this progression through a single-cell RNA landscape. We conducted single-cell RNA sequencing on three normal colonic tissues and fifteen SLs (including HPs, SSLs, SSLD, and TSAs) and integrated these data with datasets containing tumor samples. We identified three invasive malignant epithelial cell subtypes related to CRC progression: SLC1, SLC2, and tumor cell. SLC1, specific to SSLs, is involved in cell proliferation and shows a continuum of malignancy in gene expression. TSA-specific SLC2 exhibited FOXQ1 upregulation and active EMT, indicating invasiveness. The trajectory analysis showed that HPs do not progress to cancer, and different SL types are linked to the MSI status of advanced CRCs. We validated molecular drivers in premalignant lesions and later carcinogenesis. In the tumor microenvironment, CAF and pre-CAF fibroblast subtypes associated with progression were identified. During the premalignant stage, SLC1 triggered CD8+ T cell responses, while at the advanced stage, CAFs promoted tumor invasion and metastasis via FN1-CD44, influencing tumor progression and the treatment response. Our findings highlight transcriptional changes across serrated pathway stages, aiding in early CRC diagnosis and treatment.

Identification and Verification of Potential Markers Related to Myocardial Fibrosis by Bioinformatics Analysis.

Mounting evidence indicates that myocardial fibrosis (MF) is frequently intertwined with immune and metabolic disorders. This comprehensive review aims to delve deeply into the crucial role of immune-related signature genes in the pathogenesis and progression of MF. This exploration holds significant importance as understanding the underlying mechanisms of MF is essential for developing effective diagnostic and therapeutic strategies. The dataset GSE9735 about myocardial fibrosis and non-fibrosis was downloaded from GEO database. Differentially expressed genes (DEGs) were identified by 'limma' package in R software. Then, the biological function of DEG was determined by gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis. XCell was used to estimate the composition pattern of matrix and immune cells. Protein-protein interaction (PPI) network was constructed based on STRING analysis software, and Hub genes were screened and functional modules were analyzed. The correlation between hub genes and immune cell subtypes was analyzed. Hub genes with |correlation coefficient|> 0.45 and p-value < 0.05 were used as characteristic biomarkers. Finally, the logistic regression model is used to verify the three markers in the training set and verification set (GSE97358 and GSE225336). A total of 635 DEGs were identified. Functional enrichment analysis shows that inflammation and immune response, extracellular matrix and structural remodeling play an important role in the pathological mechanism of MF. Immune cell infiltration analysis showed that immune cells (Plasma cells, Eosinophils, Chondrocytes and Th2 cells) significantly changed in MF pathological conditions. In PPI network analysis, IL1β, TTN, PTPRC, IGF1, ALDH1A1, CYP26A1, ALDH1A3, MYH11, CSF1R and CD80 were identified as hub genes, among which IL1β, CYP26A1 and GNG2 were regarded as immune-related characteristic markers. The AUC scores of the three biomarkers are all above 0.65, which proves that they have a good discrimination effect in MF. In this study, three immune-related genes were identified as diagnostic biomarkers of MF, which provided a new perspective for exploring the molecular mechanism of MF. This study takes a comprehensive approach to understanding the intricate relationship between myocardial fibrosis and immune metabolism. By identifying key immune-related biomarkers, this study not only reveals the molecular basis of myocardial fibrosis but also paves the way for the development of novel diagnostic tools and therapeutic strategies. These findings are critical for improving patient prognosis and may have broader implications for studying and treating other cardiovascular diseases associated with immune dysregulation.

GlycoSS: A DNA Glycosignal Sieve for Deciphering Spatially Resolved EpCAM-Specific Glycoforms.

Malignant transformation of cancer is often accompanied by aberrant glycopatterns. Epithelial-mesenchymal transition (EMT) is a crucial biological process in cancer migration and invasion, accelerating cancer deterioration. High-precision analysis of protein-glycan spatial profiling in the EMT process is essential for elucidating glycosylation functions and cancer progression. However, the diversity of glycans in composition and conformation complicates their spatial analysis. Here, we develop a DNA glycosignal sieve (GlycoSS) visualization platform for screening glycoform expression with a protein spatial dimension. GlycoSS utilizes protein-anchored DNA nanoscanners of distinct lengths to control glycosignal readout, enabling protein-glycan distance modulations, and simultaneously orthogonally amplify glycoform output through signal amplification by an exchange reaction. Using GlycoSS, we screened EpCAM-specific hypoglycosylated glycoform signals in different breast cancer cell subtypes, especially characterizing the spatial distribution of glycans on the MCF-7 cell surface. Considering that the EpCAM-specific N-glycan dysregulation in EMT is pivotal, GlycoSS revealed dynamic glycan fluctuations during IGF-1-induced EMT, revealing that the N-glycans were positively associated with tumor malignancy and metastasis. GlycoSS is anticipated to accelerate the identification of aberrant N-glycosylation in tumor progression, advancing systemic glycobiology insights. Notably, GlycoSS is capable of analyzing diverse glycoprotein profiles, offering additional dimensions into the role of glycoprotein nanoenvironments in regulating membrane protein function.

Androgen-ablative therapies inducing CXCL8 regulates mTORC1/SREBP2-dependent cholesterol biosynthesis to support progression of androgen receptor negative prostate cancer cells.

Treatment with androgen-ablative therapies effectively inhibited androgen receptor (AR)-positive (AR+) prostate cancer (PCa) cell subtypes, but it resulted in an increase in AR-negative (AR-) PCa cell subtypes. The present study aimed to investigate the debated mechanisms responsible for the changing proportion of cell types, identifying CXCL8 as a synthetic essential effector of AR- PCa cells. AR- PCa cells were found to be susceptible to CXCL8 depletion or inhibition, which impaired their survival. Mechanistically, androgen-ablative therapies resulted in the suppression of AR signaling, leading to the upregulation of CXCL8 gene transcription. CXCL8, in turn, activated the mTORC1 pathway, which increased de novo cholesterol synthesis by activating sterol regulatory element-binding protein-2 (SREBP2). Together, these results suggested that the CXCL8-mTORC1-SREBP2 axis contributed to the exacerbation of tumorigenicity in AR- PCa cells under androgen-ablative therapies.

12527 Determining The Impact Of Dampened Oxidative Stress On Islet Function And Gene Expression During Diabetes Onset In NOD Mice

Abstract Disclosure: K.V. Coutinho: None. H. Shinde: None. J. Feduska: None. K. Burnette: None. S.O. Poole: None. H.M. Tse: None. C.S. Hunter: None. Type 1 diabetes (T1D) arises from autoimmune destruction of insulin-producing pancreatic β-cells. Revealing the mechanisms underlying T1D onset are critical for therapeutic development but not completely understood. NADPH oxidase (NOX) generates superoxide, a precursor of reactive oxygen species (ROS), to regulate cell survival, differentiation, signaling, and autoimmune responses in T1D. NOD (Non-Obese Diabetic) mice spontaneously develop T1D, featuring increased cytokine/chemokine synthesis, inflammatory responsive macrophages and T cells, and ROS. Here, we harness NOD mice deficient in NOX-derived superoxide (NOD.Ncf1m[1]J), which have significantly lower T1D incidence. However, the impacts on islet β-cells in this T1D resistant model are unknown. We assessed a time course of glucose homeostasis between 6-20 weeks (W), spanning the onset T1D in NOD mice. Surprisingly, comparative glucose tolerance testing (GTT) in NOD.Ncf1m[1]J mice revealed increased glucose intolerance at 10W with no differences at 6, 14, and 20W as compared to NOD. Insulin tolerance was similar between the two models at all stages examined. This suggests superoxide production is necessary for β-cell function, despite that NOD.Ncf1m[1]J mice are T1D resistant. To understand islet transcriptomic changes between NOD and NOD.Ncf1m[1]J mice, we conducted comparative bulk islet RNA sequencing from 6 to 20W. Principal component analysis was applied to ensure differences between genotypes at each timepoint. Quality control methods within the Nextflow core pipeline were used to ensure sample quality. As expected, NOD.Ncf1m[1]J mice had decreased Ncf1 and immune response-related mRNA levels. Expression of T-cell exhaustion (Pdcd1, Lag3, and Tigit) genes remained level in NOD mice, but had significantly lower expression at 6W in NOD.Ncf1m[1]J mice. Interestingly, Ctla4, encoding a gene product that inhibits T-cell function, was upregulated at 14W in NOD.Ncf1m[1]J mice. Furthermore, GO analysis of downregulated NOD.Ncf1m[1]J gene sets include T-cell activation, response to IFN-β and IFN-γ at 6W, and decreased cytokine production at 20W. Supporting the dysregulation of β-cell function, Bace2, encoding a protease that limits β-cell function and mass, was upregulated with NOD. Ncf1m[1]J. Additionally, 14W β-cell function gene expression was decreased in the NOD.Ncf1m[1]J mice, including Slc2a2, which encodes the β-cell GLUT2 glucose transporter, and Glp1r, which is involved in preserving β-cell mass and function. Taken together, NOX-derived superoxide, whose loss decreases T1D incidence, may also dampen β-cell gene expression and function. Future directions will include comparative NOD and NOD.Ncf1m[1]J single cell transcriptomics to determine cell type specific transcriptional signatures between islet and immune cell subtypes. Presentation: 6/2/2024