Cell Phenotype Research Articles

Association of B cells and the risk of Esophageal cancer: a bidirectional two-sample mendelian randomization study.

Currently, research on the role of B cells in esophageal cancer (EC) is limited, and existing studies on their impact are controversial. Therefore, this study was conducted to elucidate the complex causal relationship between B cells and EC, expand the understanding of esophageal cancer immunology. Bidirectional two-sample Mendelian randomization (MR) was performed to assess the causal relationships between 190 B cell phenotypes and EC. To complement the MR analysis, Bayesian Weighted Mendelian Randomization (BWMR) was employed, and sensitivity analyses were conducted to evaluate the robustness of the findings. Positive results were further validated in independent cohorts of esophageal cancer studies. In addition, RNA sequencing (RNA-seq) data from The Cancer Genome Atlas (TCGA) were utilized for validation, incorporating B cell-related gene expression analysis and functional enrichment analysis to support the MR findings. In the primary analysis, significant causal relationships were observed between 5 B cell types and the risk of EC; the onset of EC was causally linked to 3 B cell phenotypes. Validation in other cohorts revealed that 4 outcomes aligned with the primary analysis, included were CD19 on IgD + CD38-, CD20 on IgD- CD27-, CD20 on IgD- CD38br, and CD38 on PB/PC. Further validation using RNA-seq data showed that CD38 mRNA was significantly overexpressed in EC tissues, whereas CD19 and MS4A1 mRNA levels did not differ significantly between tumor and normal tissues. Functional enrichment analysis revealed that CD19, MS4A1, and CD38 are involved in multiple tumor-related immune pathways, suggesting their pivotal role in regulating the tumor immune microenvironment. Our study suggests a potential connection between B cell phenotypes and EC through bidirectional two-sample MR combined with BWMR analysis, providing a preliminary basis for future research.

NO classifier prediction of anti neuroinflammatory agents using text mining of 3D molecular fingerprints.

CNS Drug discovery has been challenging due to the lack of clarity on CNS diseases' basic biological and pathological mechanisms. Despite the difficulty, some CNS drugs have been developed based on phenotypic effects. Herein, we propose a phenotype-structure relationship model, which predicts an anti-neuroinflammatory potency based on 3D molecular structures of the phenotype-active or inactive compounds without specifying targets. For this chemo-centric study, a predictive model of the nitric oxide (NO) inhibitory potency in hyper-activated microglia is built from the 548 agents, which were collected from 95 research articles (28 substructures consisting of natural products and synthetic scaffolds) and doubly externally validated by the agents of 9 research articles as third set. 3D Structures (multi-conformer ensemble) of every agent were encoded into the E3FP molecular fingerprint of the Keiser group as a 3D molecular representation. The location information of the molecular fingerprints could be learned and validated to classify the inhibitory potency of compounds (IC50 cut-off between the active and inactive: 37.1 µM): (1) multi-layer perceptron (MLP) (AUC-CV: 0.997, AUC-Test: 0.992), (2) recurrent neural network (RNN) (AUC-CV: 0.999, AUC-Test: 0.995), and (3) convolutional neural network (CNN) (AUC-CV: 0.998, AUC-Test: 0.994). The high performance of these models was compared with that of four classical machine classification models (Logistic, Ridge, Lasso, and Naïve Bayes). We named the binary classification models NO-Classifier. Independent test set validation and decision region analysis of the independent test set doubly demonstrated NO-Classifier effectively discerned the anti-inflammatory potency of testing compounds in inflammatory cell phenotype with the webserver in https://no-classifier.onrender.com.

Microglial and Macrophage Plasticity and Regional Cerebral Blood Flow in the Prenatal Brain and Gut Under Vagus Nerve Stimulation.

An intricate relationship exists between the vagus nerve and systemic immune cell regulation, specifically during fetal development. Little is known about the connection between the vagus nerve and the brain's regional circulatory control. In this chapter, we present a methodology for studying the impact of vagus nerve signaling on these connections in the developing fetus using the sheep model for human fetal physiology. First, we present the protocol to study the connection between the vagus nerve physiology and the regional cerebral blood flow (rCBF). Next, we detail the protocol for measuring how vagal signaling alters microglial cell plasticity in gut and brain. In previous work, our team showed that vagotomy results in amplified redistribution of rCBF toward subcortical structures in the fetal brain. Conversely, efferent VNS reduces rCBF to cortical structures while afferent VNS diminishes the rise of rCBF to subcortical structures (independent of cortical rCBF) when compared to controls in the fetal brain. Additionally, our team showed that Iba-1 expression, a marker for microglial cellular signaling activation, rises in a dose-dependent relationship with systemic inflammatory activation in the setting of vagotomy. The findings support existing preclinical and clinical evidence in adult human physiology that vagotomy is neuroprotective for neurodegenerative diseases such as Parkinson's likely via a glial cell-mediated mechanism. Vagus nerve stimulation (VNS) has also been shown to alter rCBF patterns in adults with treatment-resistant depression, underscoring the importance of further investigation of the relationship between the vagus nerve and rCBF as early as in utero. Together, the body of evidence emphasizes that the vagal pathway is an important player in the programming of microglial cell phenotypes within the developing brain. Further study is needed to better understand the significance of these relationships for the development and treatment of early susceptibility to neuroinflammatory and neurodegenerative disorders in later life. Therefore, we present a methodology for assessing rCBF and morphometric features of microglial and macrophage cell activation to allow future teams to expand on the existing body of work and further examine these relationships at a cellular and systems' levels.

Role of R-spondin 2 on osteogenic differentiation of bone marrow mesenchymal stem cells and bone metabolism in ovariectomized mice

To investigate the effects of R-spondin 2 (Rspo2) on the osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) and bone mineral content in ovariectomized mice. BMSCs were extracted from the bone marrow of the long bones of 7 4-week-old female C57BL/6 mice using whole bone marrow culture and passaged. After the cell phenotype was identified by flow cytometry, the 3rd generation cells were co-cultured with 10, 20, 40, 80, and 100 nmol/L Rspo2. Then, the cell activity and proliferative capacity were determined by cell counting kit 8 (CCK-8), and the intervention concentration of Rspo2 was screened for the subsequent experiments. The osteogenic differentiation ability of BMSCs was detected by alkaline phosphatase (ALP) staining, and the mRNA levels of osteogenesis-related genes [RUNX family transcription factor 2 (Runx2), collagen type Ⅰ alpha 1 (Col1), osteocalcin (OCN)] were detected by real-time fluorescence quantitative PCR (RT-qPCR). In addition, 18 10-week-old female C57BL/6 mice were randomly divided into sham operation group (sham group), ovariectomy group (OVX group), and OVX+Rspo2-intervention group (OVX+Rspo2 group), with 6 mice in each group. The sham group only underwent bilateral back incision and suturing, while the other two groups established osteoporosis mouse models by bilateral ovarian castration. Then, the mice were given a weekly intraperitoneal Rspo2 (1 mg/kg) treatment in OVX+Rspo2 group and saline at the same dosage in sham group and OVX group. After 12 weeks of treatment, the body mass and uterus mass of the mice were weighed in the 3 groups to assess whether the OVX model was successfully prepared; the tibia bones were stained with HE and immunohistochemistry staining to observe the changes in tibial bone mass and the expression level of Runx2 protein in the bone tissues. Blood was collected to detect the expressions of bone metabolism markers [ALP, OCN, type Ⅰ procollagen amino-terminal peptide (PINP)] and bone resorption marker [β-collagen degradation product (β-CTX)] by ELISA assay. Micro-CT was used to detect the bone microstructure changes in the tibia, and three-dimensional histomorphometric analyses were performed to analyze the trabeculae thickness (Tb.Th), trabeculae number (Tb.N), trabeculae separation (Tb.Sp), and bone volume fraction (BV/TV). CCK-8 assay showed that Rspo2 concentrations below 80 nmol/L were not cytotoxic ( P>0.05), and the cell viability of 20 nmol/L Rspo2 group was significantly higher than that of the control group ( P<0.05). Based on the above results, 10, 20, and 40 nmol/L Rspo2 were selected for subsequent experiments. ALP staining showed that the positive cell area of each concentration of Rspo2 group was significantly larger than that of the control group ( P<0.05), with the highest showed in the 20 nmol/L Rspo2 group. The expression levels of the osteogenesis-related genes (Runx2, Col1, OCN) significantly increased, and the differences were significant between Rspo2 groups and control group ( P<0.05) except for Runx2 in the 40 nmol/L Rspo2 group. In animal experiments, all groups of mice survived until the completion of the experiment, and the results of the body mass and uterus mass after 12 weeks of treatment showed that the OVX model was successfully prepared. Histological and immunohistochemical staining showed that the sparseness and connectivity of bone trabecula and the expression of Runx2 in the OVX group were lower than those in the sham group, whereas they were reversed in the OVX+Rspo2 group after treatment with Rspo2, and the differences were significant ( P<0.05). ELISA assay showed that compared with the sham group, the serum bone metabolism markers in OVX group had an increase in ALP and a decrease in PINP ( P<0.05). After Rspo2 intervention, PINP expression significantly reversed and increased, with significant differences compared to the sham group and OVX group ( P<0.05). The bone resorption marker (β-CTX) was significantly higher in the OVX group than in the sham group ( P<0.05), and it was significantly decreased in the OVX+Rspo2 group when compared with the OVX group ( P<0.05). Compared with the sham group, Tb.Th, Tb.N, and BV/TV significantly decreased in the OVX group, while Tb.Sp significantly increased ( P<0.05); after Rspo2 intervention, all of the above indexes significantly improved in the OVX+Rspo2 group ( P<0.05) except Tb.Th. Rspo2 promotes differentiation of BMSCs to osteoblasts, ameliorates osteoporosis due to estrogen deficiency, and promotes bone formation in mice.

VI-VS: calibrated identification of feature dependencies in single-cell multiomics.

Unveiling functional relationships between various molecular cell phenotypes from data using machine learning models is a key promise of multiomics. Existing methods either use flexible but hard-to-interpret models or simpler, misspecified models. VI-VS(Variational Inference for Variable Selection) balances flexibility and interpretability to identify relevant feature relationships in multiomic data. It uses deep generative models to identify conditionally dependent features, with false discovery rate control. VI-VS is available as an open-source Python package, providing a robust solution to identify features more likely representing genuine causal relationships.

Peripheral CD4+ T cell phenotype and brain microglial activation associated with cognitive heterogeneity in aged rats.

Cognitive decline is a critical hallmark of brain aging. Although aging is a natural process, there is significant heterogeneity in cognition levels among individuals; however, the underlying mechanisms remain uncertain. In our study, we classified aged male Sprague‒Dawley rats into aged cognition-unimpaired (AU) group and aged cognition-impaired (AI) group by using an attentional set-shifting task. The transcriptome sequencing results of medial prefrontal cortex (mPFC) demonstrated significant differences in microglial activation and inflammatory response pathways between the two groups. Specifically, compared to AU rats, AI rats exhibited a greater presence of CD86-positive microglia and major histocompatibility complex class II (MHC-II)-positive microglia, along with elevated inflammatory molecules, in mPFC. Conversely, AI rats exhibited a reduction in the percentage of microglia expressing CD200R and the anti-inflammatory molecules Arg-1 and TGF-β. Additionally, peripheral blood analysis of AI rats demonstrated elevated levels of Th17 and Th1 cells, along with proinflammatory molecules; however, decreased levels of Treg cells, along with anti-inflammatory molecules, were observed in AI rats. Our research suggested that peripheral Th17/Treg cells and central microglial activation were associated with cognitive heterogeneity in aged rats. These findings may provide a new target for healthy aging.

Procaine regulates the STAT3/CCL5 axis and inhibits microglia M1 polarization to alleviate CFA rats pain behavior.

Neuropathic pain (NP) caused by sciatic nerve injury can significantly impact the quality of life of patients. The M1 phenotype of microglia has been reported to promote the progression of NP. Procaine is a lipid-soluble local anesthetic drug that exerts narcotic analgesic effects. Nevertheless, the detailed effect of procaine in NP is not clear. In order to explore the role of procaine in the polarization of NP microglia, HAPI cells were exposed to LPS to polarize into M1 type. In addition, the number of the M1 phenotype of HAPI cells was assessed using flow cytometry. The binding site between CCL5 and STAT3 was explored using the dual luciferase assay. Furthermore, in vivo experiments were applied for testing the impact of procaine on NP.LPS significantly inhibited HAPI cell viability, which was reversed by procaine. Consistently, procaine alleviated LPS-induced upregulation of inflammatory factors. Additionally, it significantly inhibited HAPI cells M1 polarization induced by LPS. Meanwhile, overexpression of STAT3 was able to promote HAPI cells M1 polarization through binding with the CCL5 promoter region and activating the PI3K/Akt signaling. Procaine could alleviate the painful behavior of complete Freund's adjuvant (CFA) rats by modulating the STAT3/CCL5 axis and inhibiting microglia M1 polarization. In conclusion, procaine alleviated the painful behavior of CFA rats via regulating the STAT3/CCL5 axis and inhibiting microglia M1 polarization. Hence, the research might provide a novel agent for NP treatment.Significance statement Neuropathic pain (NP) refers to pain caused by damage to the somatosensory system, which can be caused by brain or spinal cord injury and have a serious impact on the patient's quality of life. The M1 phenotype of microglia plays a crucial role in promoting the progression of NP. In this study, we investigated the specific mechanism of local anesthetic procaine in improving NP by inhibiting microglia polarization towards M1 type. Our findings may provide a new drug for NP treatment.

MicroRNA 223 Enhances ABCA1 Protein Stability and Supports Efflux in Cholesterol-Burdened Macrophages.

Macrophages are present in all vertebrates as part of the innate immune system, which protects from pathogens and scavenges sterol rich, cellular debris and modified lipoproteins. Thus, resident macrophages are prone to excessive levels of intracellular cholesterol esters. Intramacrophage cholesterol esters can efflux via cell surface transporters, ABCA1 and ABCG1, to lipoprotein carriers such as apo-AI and HDL. Systemically, Apo-AI and HDL facilitate trafficking of cholesterol back to the liver, in a process called reverse cholesterol transport. Impaired macrophage cholesterol efflux is a primary factor in the etiology of atherosclerosis. We hypothesized that microRNA 223 (miR-223) regulated macrophage LDL metabolism, due to predicted binding to Sp1 and Sp3 mRNA, transcriptional regulators of ABCA1 expression. Primary mouse (WT, miR-223 KO) macrophages were loaded with acetylated LDL and stimulated with LPS to form an inflammatory foam cell phenotype. miR-223 KO foam cells demonstrated impaired efflux to both apo-AI and HDL. While transcriptional regulation was intact in miR-223 KO foam cells, ABCA1 protein degradation was greatly accelerated. Blockade of both proteasomal and lysosomal degradation pathways rescued miR-223 deficiency-mediated ABCA1 degradation to the WT levels. Our findings demonstrate that miR-223 expression in macrophages is required for maintenance of ABCA1 and ABCG1 proteins.

Natural killer cell biology and therapy in multiple myeloma: challenges and opportunities.

Despite therapeutic advancements, multiple myeloma (MM) remains incurable. NK cells have emerged as a promising option for the treatment of MM. NK cells are heterogenous and typically classified based on the relative expression of their surface markers (e.g., CD56 and CD16a). These cells elicit an antitumor response in the presence of low mutational burden and without neoantigen presentation via germline-encoded activating and inhibitory receptors that identify the markers of transformation present on the MM cells. Higher NK cell activity is associated with improved survival and prognosis, whereas lower activity is associated with advanced clinical stage and disease progression in MM. Moreover, not all NK cell phenotypes contribute equally toward the anti-MM effect; higher proportions of certain NK cell phenotypes result in better outcomes. In MM, the proportion, phenotype, and function of NK cells are drastically varied between different disease stages; this is further influenced by the bone marrow microenvironment, proportion of activating and inhibitory receptors on NK cells, expression of homing receptors, and bone marrow hypoxia. Antimyeloma therapies, such as autologous stem cell transplant, immunomodulation, proteasome inhibition, and checkpoint inhibition, further modulate the NK cell landscape in the patients. Thus, NK cells can naturally work in tandem with anti-MM therapies and be strategically modulated for improved anti-MM effect. This review article describes immunotypic and phenotypic differences in NK cells along with the functional changes in homeostatic and malignant states and provides expert insights on strategies to harness the potential of NK cells for improving outcomes in MM.

LINC01614 Promotes Oral Squamous Cell Carcinoma by Regulating FOXC1

Background: Long non-coding RNAs (lncRNAs) are pivotal mediators during the development of carcinomas; however, it remains to be investigated whether lncRNAs are implicated in oral squamous cell carcinoma (OSCC). Methods: In this study, quantitative real-time PCR was conducted for detecting the expression of LINC01614 in OSCC cell lines. The biological functions of LINC01614 were assessed by loss- and gain-of-function experiments conducted both in vivo and in vitro. Cellular proliferation, migration, and invasion were investigated herein, and dual luciferase reporter assays were additionally performed to explore the relationships among LINC01614, miR-138-5p, and Forkhead box C1 (FOXC1). Results: The research presented herein revealed that OSCC cells express high levels of LINC01614. Functional experiments employing cellular and animal models demonstrated that LINC01614 knockdown repressed the malignant phenotypes of OSCC cells, including their growth, invasiveness, and migration. Further investigation revealed that LINC01614 absorbs miR-138-5p miRNA by functioning as a competing endogenous RNA to downregulate the abundance of FOXC1. Conclusions: The findings revealed that LINC01614 contributes to the progression of OSCC by targeting the FOXC1 signaling pathway. The study provides insights into a novel mechanistic process to regulate the development of OSCC, and established a possible target for the therapeutic management of OSCC.

Rediscovering publicly available single-cell data with the DISCO platform.

Single-cell RNA sequencing (scRNA-seq) has emerged as the key technique for studying transcriptomics at the single-cell level. In our previous work, we presented the DISCO database (https://www.immunesinglecell.org/) that integrates publicly available human scRNA-seq data. We now introduce an enhanced version of DISCO, which has expanded fourfold to include >100 million cells from >17 thousand samples. It provides uniformly realigned read count tables, curated metadata, integrated tissue and phenotype specific atlases, and harmonized cell type annotations. It also hosts a single-cell enhanced knowledgebase of cell type ontology and gene signatures relating to cell types and phenotypes. Lastly, it offers a suite of tools for data retrieval, integration, annotation, and mapping, allowing users to construct customized atlases and perform integrated analysis with their own data. These tools are also available in a standalone R package for offline analysis.

Impact of Psychological Stressors on Natural Killer Cell Function: A Comprehensive Analysis based on Stressor Type, Duration, Intensity, and Species.

Patients with natural killer (NK) cell deficiency or dysfunction are more susceptible to infections by Herpesviridae viruses, herpesvirus-related cancers, and macrophage activation syndromes. This review summarizes research on NK cell dysfunction following psychological stress, focusing on stressor type, duration, age of exposure, and species studied. Psychological stressors negatively affect NK cell activity (NKCA) across species. Prolonged stress leads to more significant decreases in NK cell number and function, with rehabilitation efforts proving ineffective in reversing these effects. Early life and prolonged stress exposure particularly increases the risk of infections and cancer due to impaired NKCA. The review also highlights that stress impacts males and females differently, with females exhibiting a more immunosuppressed NK cell phenotype. Notably, mice respond differently compared to humans and other animals, making them unsuitable for NK cell stress-related studies. Most studies measured NKCA using cytolytic assays against K-562 or YAC-1 cells. Although the exact mechanisms of NK cell dysfunction under stress remain unclear, potential causes include reduced release of secretory lysosomes with perforin or granzyme, impaired NK cell synapse formation, decreased expression of synapse-related molecules like CD2 or LFA-1 (CD11a), altered activating receptor expression, and dysregulated signaling pathways, such as decreased Erk1/2 phosphorylation and NFkB signaling. These mechanisms are not mutually exclusive, and future research is needed to clarify these pathways and develop therapeutic interventions for stress-induced immune dysregulation.

Association between immune cells and urticaria: a bidirectional Mendelian randomization study

Urticaria is characterized by transient itchy symptoms on the skin, usually accompanied by swelling, which is caused by mast cell activation leading to increased vascular permeability and dilation of the dermis. Urticaria involves recurrent activation of mast cells, T cells, eosinophils, and other immune cells around lesioned venules, with complex regulatory systems affecting mast cell functions, potentially contributing to urticaria pathogenesis. The direct causal relationship between immune cells and urticaria is currently unclear. To address this, our study utilized a bidirectional Mendelian randomization analysis, employing instrumental variables (IVs) associated with immune cells and urticaria, to investigate this causal relationship. First, by utilizing Genome-wide Association Study (GWAS) data, we identified 31 immunophenotypes associated with urticaria risk, with 18 increasing and 13 decreasing the risk. Through rigorous criteria, we identified 4 immunophenotypes that have a strong causal relationship with urticaria. Notably, HLA DR+ CD4+AC, CD45 on CD8br, and HLA DR on plasmacytoid dendritic cells were associated with an increased risk, while CD8dim NKT %lymphocyte was identified as a protective factor. Sensitivity analyses, including the MR-Egger intercept test, scatter plots, funnel plots, and leave-one-out analysis, supported the robustness of the findings. Reverse MR analysis suggested an inverse causal effect of urticaria on CD8dim NKT %lymphocyte, reinforcing the potential bidirectional nature of the relationship between urticaria and immune cell phenotypes. Our research substantiates the bidirectional causal relationship between immune cells and urticaria, thus benefiting for urticaria-targeted therapy development.

Evaluating the causal association between circulating plasma proteins, 731 immune cell phenotypes, and atopic dermatitis: A mediation Mendelian randomization study.

Atopic dermatitis (AD) is a chronic inflammatory skin disease characterized by eczematous lesions and severe itching. However, its pathogenesis has not yet been fully elucidated. The aim of this study was to investigate the causal relationship between plasma proteins and AD, as well as to identify and quantify the potential roles of immune cell phenotypes as mediators. We utilized summary-level data from genome-wide association studies and conducted a two-sample Mendelian randomization (MR) analysis involving 4907 circulating plasma proteins, 731 immune cell phenotypes, and AD. Initially, we conducted bidirectional univariate MR analyses to forecast causal effects linking circulating plasma proteins and AD. Subsequently, we employed a two-step MR analysis to scrutinize the immune cell phenotypes that could mediate these effects. The inverse variance weighted was the main method employed for MR analysis, while the Cochran's Q test and MR-Egger intercept test were used to assess the presence of heterogeneity and pleiotropy, respectively. We then determined whether our results could be influenced by individual single-nucleotide polymorphisms using the "leave-one-out" test. Positive correlations were observed between KRT1, IL18R1, and SEMA6A and the risk of AD, whereas BDH2, ADAMTS3, ANKRD1, TIAM1, MID2, and IFNA16 all showed negative correlations with the risk of AD. Mediation analysis indicated that CD8 on CM CD8br cells acted as a mediator between IFNA16 and AD, with a mediation effect proportion of 11.2%. In addition, sensitivity analyses did not reveal significant heterogeneity or level pleiotropy. Our findings indicated the presence of a one-way causal relationship between the circulating plasma protein IFNA16 and AD. This study also explored immune cell phenotypes that may serve as mediators, offering novel insights into the etiology, pathogenesis, and potential clinical interventions in AD. Nevertheless, these findings need to be validated by clinical and laboratory studies.

Abstract B036: Pathway repression in progression on osimertinib therapy in NSCLC via a comprehensive genomic and epigenomic circulating tumor DNA (ctDNA) assay

Abstract Background: Osimertinib is currently the most common precision therapy for patients with non- small cell lung cancer (NSCLC) with EGFR mutations. While it prolongs progression-free survival, resistance eventually develops in many, leading to disease progression. Somatic NGS has identified resistance mutations in the EGFR pathway and other oncogenes, but these account for only 40-45% of cases, suggesting non-genetic factors, such as epigenetic changes, may be involved. This study investigates the epigenetic landscape at diagnosis and progression on first- line osimertinib, focusing on cases with and without canonical resistance mutations. Methods: We defined two cohorts from GuardantINFORM, a clinico-genomics database, restricting to only patients with a test on the Guardant Infinity platform, a comprehensive blood-based NGS assay, that analyzes DNA sequence variants and performs extensive methylation profiling (∼15Mb) in &amp;gt;20,000 differentially methylated regions (DMRs) assessed for sample-specific methylation patterns. (A) Diagnosis Cohort: Comprised of 34 NSCLC patients with blood samples collected within six days prior to the initiation of first-line osimertinib therapy. (B) Progression Cohort: Comprised of 36 patients who were on first-line osimertinib therapy, with blood samples collected within 60 days of discontinuation. The progression cohort was further stratified based on the presence or absence of known resistance mutations identified through somatic variant analysis by Guardant360. Gene set enrichment analysis was conducted on these methylation profiles using the Reactome database to define pathway-level differences between the cohorts. Results: Pathway enrichment analysis in the diagnosis and progression cohorts found expected biological processes in NSCLC such as extracellular matrix organization, G- protein coupled receptor (GPCR) signaling, and FGFR pathways. Additionally, the progression cohort lacking canonical resistance mutations (n=23) exhibited unique enrichment in two specific pathways: Glucose-dependent Insulinotropic Polypeptide and Physiological Factors. These pathways include genes such as GATA4, which has been implicated in the epithelial- mesenchymal transition (EMT)—a process associated with transforming to a small cell phenotype, a known mechanism of resistance to EGFR inhibition, including osimertinib. Conclusion: This study demonstrates the utility of epigenetic profiling via liquid biopsies in uncovering non-genetic mechanisms underlying osimertinib resistance in advanced NSCLC. Identification of unique epigenetic pathways in progressing patients without canonical resistance mutations highlights the potential for discovering novel biomarkers of resistance. Further investigation into these findings may advance our understanding of resistance mechanisms across various cancer therapies, particularly for patients without identified genomic resistance, and inform clinical decision-making and trial design. Citation Format: Aaron Hardin, Leslie Bucheit, Amar Das, Craig Eagel, Helmy Eltoukhy. Pathway repression in progression on osimertinib therapy in NSCLC via a comprehensive genomic and epigenomic circulating tumor DNA (ctDNA) assay [abstract]. In: Proceedings of the AACR Special Conference: Liquid Biopsy: From Discovery to Clinical Implementation; 2024 Nov 13-16; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(21_Suppl):Abstract nr B036.

Abstract PR005: Leveraging mass cytometry for phenotyping CTCs in SCLC liquid biopsies: Tracking therapy resistance at a personalized level

Abstract Liquid biopsies present a promising, minimally invasive procedure through which clinicians can assess and monitor disease status by interrogating circulating tumor cell (CTC) and immune cell phenotypes. In the case of Small-cell lung cancer (SCLC) – a type of lung cancer characterized by aggressive growth, dismal prognosis and limited treatment options - leveraging liquid biopsies and CTCs for evaluating disease status would be of great benefit, given that biopsies are scarce. Furthermore, SCLC CTC numbers are higher compared to other cancers, presenting a unique opportunity to utilize them for assessing SCLC therapy resistance and identifying new targetable markers. In this study, we leverage mass cytometry (i.e. CyTOF) to phenotype SCLC CTC phenotypes in longitudinal clinical specimens to assess therapy resistance at a personalized level. By using CyTOF - a multiplex cytometric approach that enables interrogation of 30-50 protein markers per single cell – we show that we are able to identify and analyze a significant number of CTCs and their heterogeneous expression profiles. To achieve this, we first optimized a CyTOF panel of ∼25-30 antibodies by using SCLC cell lines and blood specimens from healthy donors and SCLC patients. We show that by using this approach, we are able to identify and phenotype SCLC CTCs by using the combination of CD45 (negative expression) and CD56 positive expression with further validation achieved by TTF1 and SCLC subtype transcription factor expression (NEUROD1, POU2F3 and ASCL1). This approach avoids enriching for specific CTC epithelial phenotypes that express surface markers that fluctuate during epithelial- mesenchymal transition (EMT) (e.g. EpCAM, a common marker for isolating CTCs). For parallel immune profiling we assessed among others CD8/CD4 (T cells) and CD11b/HLA-DR (Myeloid-derived suppressor cells, MDSCs) expression. We then proceeded to interrogate CTC phenotypic differences in the naïve vs relapsed setting as well as dynamic changes in longitudinal blood specimens from SCLC patients by evaluating markers that describe immune suppression activity (PD-L1) and EMT status (E-Cadherin, Vimentin, MUC1, Twist and Slug). Importantly, we show that with our approach we can track in real time therapeutic targets that are currently being tested in clinical trials or have been recently FDA approved (e.g. pYAP and DLL3) and found that MUC1 and pYAP are significantly increased in CTCs detected in blood specimens of SCLC relapsed patients. Our immune profiling showed an increase of MDSC percentages and a decrease in the CD4/CD8 T cell ratio in the relapsed setting. Finally, when we used PHENOSTAMP, a previously developed reference EMT map for assessing EMT phenotypes in lung cancer clinical specimens, we observed an increase of EMT heterogeneity in CTCs in relapsed patients. Our study highlights the translational power of our approach in utilizing CyTOF for longitudinally tracking CTCs directly in SCLC patient liquid biopsies towards assessing therapy response and resistance at a personalized level. Citation Format: Loukia G Karacosta, Sayantan Bhattacharyya, Allison Stewart, Ashley Victorian, Ester F Lujan, Shafqat Ehsan, Subin Kim, Alberto Duarte, Runsheng Wang, Benedict Anchang, Jing Wang, Lauren Byers. Leveraging mass cytometry for phenotyping CTCs in SCLC liquid biopsies: Tracking therapy resistance at a personalized level [abstract]. In: Proceedings of the AACR Special Conference: Liquid Biopsy: From Discovery to Clinical Implementation; 2024 Nov 13-16; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(21_Suppl):Abstract nr PR005.

Abstract 4137500: The Histone Methyltransferase MLL1 Regulates Notch Signaling and T Cell Phenotype During Pathologic Abdominal Aortic Aneurysm Development

Objective: Abdominal aortic aneurysms (AAA) are characterized by pathological vascular remodeling and an imbalance between anti-inflammatory regulatory T H cells (Tregs) and pro-inflammatory T H 17 cells within the aortic wall. The mechanisms regulating CD4 + T H cell differentiation during AAA development remain unknown. Recently, the histone methyltransferase, mixed lineage leukemia 1 (MLL1), has been shown to influence T H cell phenotype by directly altering key transcription factors for T H 17 (RORγ) and Treg (FOXp3) differentiation in varying disease states. As such the objective of this study was to evaluate the role of MLL1 in promoting T H 17 activity within AAA development and to determine key T H 17 cell signaling pathways that are altered Methods: Single-cell sequencing was conducted on human AAAs and control tissue samples. For our murine model, C57BL/6 mice were injected with an AAV encoding a PCSK9 gain-of-function mutation and fed a saturated fat diet followed by either AngII infusion to induce AAAs (1 µg/kg/min) or saline. Mice with T-cell specific deletion of Notch1 signaling (Notch1 f/f CD4 cre+ ) or MLL1 ( Mll1 f/f CD4 cre+ ) were subjected to the AngII-induced AAA model and AAA diameters quantified. CD4+ T-cells were isolated by MACs and gene expression analyzed by qPCR as well as T-cell phenotype by flow cytometry. ChIP was used to evaluate histone 3 lysine trimethylation (H3K4me3). Results: Single-cell RNA sequencing of human aortic tissue revealed that the epigenetic enzyme MLL1 and Notch1 signaling were profoundly upregulated in human AAA CD4+ cells compared to non-aneurysmal control samples. Congruently, CD4 + T H cells isolated from the murine AngII-induced AAA model displayed a predominance of T H 17 phenotype and elevated expression of MLL1. Histone methylation was evaluated by ChIP in AngII-induced AAA CD4+ cells and demonstrated increased levels of the MLL1-mediated activation histone methylation mark, H3K4me3, on the Notch 1 promoter. Mice deficient in CD4 + T H cell Notch signaling ( Notch1 f/f CD4 cre+ ) or MLL1 ( Mll1 f/f CD4 cre+ ) subjected to the AngII-induced murine model displayed significant reduction AAA development (n=10/group, p&lt;0.05) with decreased CD4+ T H 17 cell activity and reciprocal increase in Treg expression. Conclusions: MLL1, a histone methyltransferase, regulates Notch1 signaling and T-cell differentiation in AAA development, causing pathologic inflammation. This pathway may serve as a potential therapeutic target to prevent aortic dilation.

SSX2IP promotes cell proliferation and migration in breast cancer by regulating FANCI.

Synovial sarcoma X breakpoint 2 interacting protein (SSX2IP) is expressed in various normal tissues and participates in the progression of human cancers. Nevertheless, the specific functions and underlying molecular mechanisms of SSX2IP in cancer, particularly in breast cancer, remain poorly understood. In this study, we aimed to explore the functional role of SSX2IP in breast cancer. Immunohistochemical staining, quantitative real-time PCR, and western blotting blot analysis were used to assess genes expression levels. By manipulating SSX2IP expression levels and conducting functional assays including Celigo cell counting assay or CCKCCK-8-8 assay, flow cytometry, wound healing assay, and Transwell assay, we explored the impact of SSX2IP on the malignant phenotype of breast cancer cells. Additionally, the in vivo tumor-suppressive ability of SSX2IP was investigated by tumor xenograft experiment. Our results revealed an upregulation of SSX2IP in the breast cancer. Functional assays demonstrated that SSX2IP knockdown inhibited cell proliferation and migration, induced apoptosis in vitro, as well as suppressed the tumor growth in vivo. Conversely, SSX2IP overexpression contributed to the malignant phenotype of breast cancer cells. Co-expression analysis showed that FA Complementation Group I (FANCI) was co-expressed with SSX2IP. Additionally, SSX2IP positively regulated FANCI expression and its interaction was verified by Co-IP.Co-IP. Furthermore, FANCI overexpression partially reversed the effects of SSX2IP knockdown on cell proliferation and metastasis. In summary, our findings revealed that SSX2IP contributes to the progression of breast cancer by regulating FANCI, hinting at its potential as a novel biomarker and therapeutic target for the treatment of breast cancer.

Abstract 4136905: EF-24, a curcumin analog, reverses interleukin-18-induced miR-30b or miR-342-dependent TRAF3IP2 expression, RECK suppression, and the proinflammatory phenotype of human aortic smooth muscle cells

Curcumin, a polyphenolic compound derived from the widely used spice Curcuma longa, has shown anti-atherosclerotic effects in cultured vascular cells and animal models. We previously reported that the induction of the proinflammatory molecule TRAF3IP2 (TRAF3 Interacting protein 2) or inhibition of the matrix metalloproteinase (MMP) regulator RECK (Reversion Inducing Cysteine Rich Protein with Kazal Motifs) contributes to agonist-induced proinflammatory, pro-oxidant, pro-mitogenic and pro-migratory effects in vascular smooth muscle cells. Here we hypothesized that, EF-24 ((3E,5E)-3,5-bis[2-fluorophenyl (methylene]-4piperidinone), a curcumin analog with better bioavailability and potency, reverses interleukin (IL)-18-induced TRAF3IP2 induction, RECK suppression and the proinflammatory phenotype of primary human aortic smooth muscle cells (ASMC). Exposure to recombinant human IL-18 (10 ng/ml) induced TRAF3IP2 mRNA and protein expression, but markedly suppressed RECK in a time-dependent manner. Further investigations revealed that IL-18 induced miR-30b, but suppressed miR-342, in a p38 MAPK and JNK-dependent manner. While miR-30b inhibitor blunted IL-18-induced TRAF3IP2 expression, miR-30b inhibitor and miR-342 mimic each restored RECK expression. Further, IL-18 induced ASMC migration (Boyden chamber assay) and proliferation (CyQUANT Cell Proliferation assay), and these effects were reversed by TRAF3IP2 knockdown or RECK overexpression. Our results also show that IL-18 induced MMP2 and MMP9 expression and activity, and targeting their expression inhibited IL-18-induced ASMC migration. TRAF3IP2 knockdown or RECK overexpression also reversed IL-18-induced ASMC proinflammatory phenotype switching as evidenced by the enhanced expression of the SMC markers ACTA2 and MYH11, and reduced expression of the proinflammatory markers Galectin 3, Olr1, VCAM, CCL2, IL-6, IL-8, and TNF-α. Importantly, preincubation with EF-24 markedly suppressed IL-18-induced SMC proliferation, MMP expression, migration and the proinflammatory phenotype switching. Together, these results suggest that the curcumin analog EF-24 has therapeutic potential in vascular inflammatory and proliferative diseases, including atherosclerosis, by differentially regulating TRAF3IP2 and RECK in vascular SMC.

Abstract 4118084: Targeting Monoamine Oxidase B in Dystrophic mdx Hearts Dampens Inflammation and Fibrosis

Background: Cardiomyopathy is a major cause of death for Duchenne muscular dystrophy (DMD) patients. Current treatments cannot prevent cardiac tissue remodeling. Oxidative stress, inflammation and fibrosis are early events in DMD, preceding heart dysfunction. Monoamine Oxidase B (MAOB), which forms H 2 O 2 by oxidizing amines, is overactivated in inflammatory conditions and overcomes cell antioxidant defenses, thus altering redox homeostasis and eliciting harmful effects. We showed that targeting MAOB with inhibitors (iMAOB) improves skeletal muscle function in dystrophic mice by lowering oxidative stress, and reduces inflammation in murine models of sepsis and arthritis by dampening NF-kB, a redox-sensitive transcription factor. Aim: We explore the therapeutic potential of iMAOB to alleviate cardiomyopathy using dystrophin-deficient mdx mice. We hypothesize that iMAOB treatment could dampen oxidative stress, inflammation and fibrosis in dystrophic mdx hearts by modulating the phenotype of cells that are crucial in cardiac remodeling. Methods: Three-month-old mdx mice, that already show signs of fibrosis but no cardiac dysfunction, were orally treated with iMAOB or vehicle for one month (n≥ 6). Heart ventricular mononucleated cells were obtained by enzymatic digestion. Myeloid, endothelial and fibroblast cells were isolated by cell sorting by FACS and analysed by RT-PCR. Oxidative stress, inflammation and fibrosis were measured in whole tissue by immunohistochemistry. Results: The expression of proinflammatory and profibrotic genes was markedly increased in myeloid [Interleukin (Il)-1b, Il-6, Tgf-b and Spp1, the gene coding for osteopontin], endothelial [Il-1b, Il-6, mmp2 and nos3] and cardiac fibroblast cells [Tgf-b, Spp1, Timp1 and Col1] isolated from mdx hearts, as compared to wild type mice. All these genes were significantly dampened by iMAOB treatment. In parallel, once again iMAOB treatment blunted the increase in oxidative stress, inflammation and fibrosis observed in mdx cardiac sections. Of notice, the differences were not linked to changes in the percentage of the various cell types, as they were unmodified amongst wild type, mdx and iMAOB-treated mdx hearts. Conclusions: We show that iMAOB can positively affect the phenotype of cells that are important in cardiac tissue remodeling. Our data suggest that iMAOB can be a viable therapeutic tool in DMD cardiomyopathy. As iMAOB are already in clinical use, such approach could be easily translated to patients.