Seq Analysis Research Articles

Skeletal muscle-specific Bambi deletion induces hypertrophy and oxidative switching coupling with adipocyte thermogenesis against metabolic disorders.

Skeletal muscle plays a significant role in both local and systemic energy metabolism. The current investigation aims to explore the role of the Bambi gene in skeletal muscle, focusing on its implications for muscle hypertrophy and systemic metabolism. We hypothesize that skeletal muscle-specific deletion of Bambi induces muscle hypertrophy, improves metabolic performance, and activates thermogenic adipocytes via the reprogramming of progenitor of iWAT, offering potential therapeutic strategies for metabolic syndromes. Leveraging the Chromatin immunoprecipitation (ChIP)-seq and bioinformatics analysis, Bambi gene is shown to be a direct target of HIF2α, which is further confirmed by ChIP-qPCR and promoter luciferase assay. Skeletal muscle-specific Bambi deletion led to significant muscle hypertrophy and improved metabolic parameters, even under high-fat diet conditions. This deletion induced metabolic reprogramming of stromal vascular fractions (SVFs) into thermogenic adipocytes, contributing to systemic metabolic improvements, potentially through the secretory factor. Notably, mice with skeletal muscle-specific Bambi deletion demonstrate resistance to high-fat diet-induced metabolic disorders, highlighting a potential therapeutic pathway for metabolic syndrome management. Thus, skeletal muscle-specific deletion of Bambi triggers muscle growth, enhances metabolic performance, and activates thermogenic adipocytes. These findings suggest Bambi as a novel therapeutic target for metabolic syndromes, providing new insights into the interaction between muscle hypertrophy and systemic metabolic improvement. The study underscores the potential of manipulating muscle physiology to regulate whole-body metabolism, offering a novel perspective on treating metabolic disorders.

IFN- γ signaling is required for the efficient replication of murine hepatitis virus (MHV) strain JHM in the brains of infected mice.

Neurotropic viruses are a major public health concern as they can cause encephalitis and other severe brain diseases. Many of these viruses, including flaviviruses, herpesviruses, rhabdoviruses and alphaviruses enter the brain through the olfactory neuroepithelium (ONE) in the olfactory bulbs (OB). Due to the low percentage of encephalitis that occurs following these infections, it's thought that the OBs have specialized innate immune responses to eliminate viruses. Murine hepatitis virus strain JHM (JHMV) is a model coronavirus that causes severe encephalitis in mice and can access the brain through olfactory sensory neurons. We've shown that a JHMV Mac1-mutant virus, N1347A, has decreased replication and disease in the brains of mice. Here we further show that this virus replicates poorly in the OB. However, it is unknown which innate immune factors restrict N1347A replication in the OB. RNA seq analysis of infected olfactory bulbs showed that IFNγ was upregulated in the OB while IFN- β was barely detectable at 5 days post-infection. To determine if IFN-γ restricts JHMV N1347A replication, we utilized IFN-γ and IFN-γ receptor (IFN-γR) knockout (KO) mice. Surprisingly we found that JHMV WT and N1347A replicated very poorly in the OB and whole brains of both IFN-γ and IFN-γR KO mice following intranasal infection, though survival and weight loss were unaltered. Furthermore, we determined that microglia were the primary cells producing IFN-γ during the early stages of this infection. We conclude that IFN-γ is required for the efficient replication of JHMV in the brains of infected mice.

ROS Responsive Cerium Oxide Biomimetic Nanoparticles Alleviates Calcium Oxalate Crystals Induced Kidney Injury via Suppressing Oxidative Stress and M1 Macrophage Polarization.

Emerging studies have demonstrated that M1 macrophage polarization and oxidative stress play important roles in calcium oxalate (CaOx) induced kidney injury, which leads to increased crystals deposition. ROS scavenging nanozymes and kidney-targeted nanoparticles for antioxidant drugs delivery have emerged as an arisen methodology for kidney injury therapy. However, cell membrane biomimetic-modified nanozymes as anti-inflammatory drug delivery systems for the treatment of kidney injury is rarely reported. Herein, the ROS responsive red blood cell-membrane-coated resatorvid-loaded cerium oxide nanoparticles (RBCM@CeO2/TAK-242) are constructed to suppress CaOx induced kidney injury and crystals deposition. In vitro, RBCM@CeO2/TAK-242 shows effective internalization by renal tubular epithelial cells, along with demonstrated antioxidative, anti-inflammatory, and macrophage reprogramming effects. Glyoxalate(Gly)-induced renal CaOx crystals mouse model is established, RBCM@CeO2/TAK-242 shows excellent injured kidney targeting and biosafety, and could effectively suppress CaOx induced kidney injury and crystals deposition. RBCM@CeO2/TAK-242 has a dual protective effect by both inhibiting oxidative stress and modulating macrophage polarization in vivo. In addition, RNA seq analysis reveals that RBCM@CeO2/TAK-242 protects against CaOx induced kidney injury via suppressing the TLR4/NF-κB pathway. This study provides an innovative strategy for RBCM@CeO2/TAK-242 as injured kidney targeting and dual protective effects for the treatment of CaOx induced kidney injury and crystals deposition.

Essential blood molecular signature for progression of sepsis-induced acute lung injury: Integrated bioinformatic, single-cell RNA Seq and machine learning analysis.

In this study, we aimed to identify an essential blood molecular signature for chacterizing the progression of sepsis-induced acute lung injury using integrated bioinformatic and machine learning analysis. The results showed that a total of 88 functionally related ALI-associated hub genes in sepsis were identified by MCODE analysis and they were enriched in infection and inflammtory responses, lung and cardiovascular disease pathways. These hub genes stratified ALI-sepsis and sepsis and further stratified two subtypes of sepsis-ALI with differential ALI scores, hub gene expression patterns, and levels of immune cells. A seven-gene signature including TNFRSF1A, NFKB1, FCGR2A, NFE2L2, ICAM1 and SOCS3 and PDCD1 was derived from the hub genes. These genes were significantly implicated in immune and metabolism pathways. They were expressed in six circulatory immune cells based on analysis of a single cell RNA sequencing dataset. Furthermore, the seven-gene signature was corrobarated using by integrating 12 machine learning algorithms. A premium three-gene signature NFE2L2, FCGR2A and PDCD1 for differentiating ALI-sepsis from sepsis were also derived from the seven-gene signature based on analysis of the seven core hub genes by the machine learning algorithms. Furthermore, the expressions of hub genes were verified in sepsis mice models. Therefore, our study provided an avenue to develop a molecular tool for identify and characterize progression of acute lung injury associated with sepsis.

Complementary and additive functions of TRα and TRβ during intestinal remodeling as revealed by ChIP-Seq analysis on wild type and TR knockout animals

Intestinal structure is drastically changed from fetal to adult form during postembryonic development, a period around birth in mammals. This process is regulated by thyroid hormone (T3) via its receptors, T3 receptor (TR) α and TRβ during anuran metamorphosis. Here, we used intestinal remodeling during Xenopus tropicalis metamorphosis, which serves as a model for human postembryonic development, to identify TR-bound genes and determine the relative contribution to target gene binding by TRα and TRβ. We first examined the localization of TRα and TRβ mRNA during metamorphosis in Xenopus tropicalis and found that TRα was broadly expressed in the intestinal tissues from premetamorphosis to the end of metamorphosis, while TRβ was expressed at low levels during premetamorphosis but was upregulated at the climax of metamorphosis when intestinal stem cells are formed and proliferate. Interestingly, both TR genes were co-expressed in different cell types, including stem cells. Chromatin immunoprecipitation (ChIP)-seq analyses of the intestine from wild type, TRα- or TRβ-knockout premetamorphic tadpoles treated with or without T3 for 18 h identified many TR-bound genes and revealed the effects of individual TR knockout on the binding of target genes by TR. We found that individual TR knockout reduced both the number of TR-bound genes and the extent of TR binding to target genes with TRα knockout had a much more dramatic effect than TRβ knockout. On the other hand, the TR-bound genes were largely common among the three genotypes. These findings suggest that both TRα and TRβ contribute to target binding with TRα having a bigger contribution in premetamorphic intestine.

Gut microbiota modulation by L-Fucose as a strategy to alleviate Ochratoxin A toxicity on primordial follicle formation

In this study, we investigated the potential benefits of L-Fucose administration to pregnant mice exposed to Ochratoxin A (OTA), a widespread mycotoxin, producing ovarian damage in offspring. The results showed that administration of 3.5 μg/d OTA induced alterations in intestinal tissues and gut microbiota of pregnant mice, leading to heightened local and systemic inflammation. This inflammatory affected the ovaries of their 3 dpp offspring, in which elevated levels of LPS and ROS were found associated to significant decreased oocyte count and impaired primordial follicle assembly. Moreover, mRNA-Seq analysis showed significant changes in ovarian transcriptomes linked to various GO terms and KEGG pathways, notably ferroptosis, a recognized form of cell death observed. Interestingly, administration of 0.3 g/kg b. w. L-Fucose following OTA exposure mitigated these effects on intestinal tissues and gut microbiota in mothers and on the offspring's ovaries. Similar benefits were obtained by gut microbiota transplantation from L-Fucose-treated pregnant females into OTA-exposed mothers. These findings suggest that inflammatory impact of OTA on maternal intestine/gut can pass to the fetus causing offspring ovary defects and support the use of L-Fucose as adjuvant to counteract the adverse effects of mycotoxins on the gut microbiota, particularly reference to those affecting reproductive organs.

Abstract 4137693: CC-Chemokine Receptor 2 Inhibition Prevents Monocyte/Macrophages Recruitment in Abdominal Aortic Aneurysms

Intro: Abdominal aortic aneurysm (AAA) is a prevalent condition in the elderly, with a significant risk of mortality if it ruptures. Despite this, there is currently no effective medical therapy to prevent AAA growth and rupture. The monocyte chemoattractant protein (MCP-1) / C-C chemokine receptor type 2 (CCR2) axis appears to play a role in AAA development. We demonstrated the potential of CCR2 as a theranostic marker for predicting AAA rupture, along with the effectiveness of a CCR2 inhibitor (RS-504393) in mitigating rupture. We further hypothesized that CCR2 inhibition can impact monocytes/macrophages trafficking in aortic tissue. Mehtods: Sprague-Dawley rats (N=54) underwent intraluminal aortic exposure to porcine pancreatic elastase (PPE) to induce aneurysmal degeneration. To stimulate AAA rupture, rats also recieved β-aminopropionitrile (BAPN). After 3 days of PPE exposure, a group of rats with AAA were administered a CCR2 inhibitor (CCR2i) either daily until the end of the study (CCR2i Full), or discontinued at day 5 (CCR2i Short), while the remaining rats proceeded without intervention (Fig1A). Ultrasound (12MHz) was performed at 7&14 days to assess diameter, while another cohort of animals were euthanized at 6 days, prior to rupture, for single-cell RNA sequencing analysis. The relative content of inflammatory cells within the aortic tissue was analyzed. Results: Both CCR2i Short and CCR2i Full treated, exhibited a significant reduction in AAA diameter at day 7 and 14 compared to controls (p<0.05; Fig1B&C). Notably, rats treated with CCR2i Full did not experience any AAA ruptures, while controls had a 56% risk of rupture. In contrast, CCR2i Short delayed the occurrence of ruptures without reducing the rupture rate (p<0.01; Fig1D). Single-cell RNA seq analysis of aortic tissue revealed that CCR2 inhibition led to a decrease in the number of recruited monocytes/macrophages. Cell recruitment was also relative to the duration of CCR2i treatment (Fig1E-G). Conclusions: Our study findings highlight the impact of CCR2-targeting in reducing AAA growth and the risk of rupture. Interestingly, length of CCR2i was a crucial variable for AAA rupture prevention. Furthermore, treatment length impacted the relative recruitment of monocytes/macrophages to AAA tissue, highlighting the role these cells in AAA disease pathology. Future analysis of CCR2+ inflammatory cell subgroups may help elucidate how CCR2 mechanistically modulates AAA progression and rupture risk.

TMIC-78. CHEMOTHERAPY INDUCED IMMUNOGENIC RESPONSE IN GLIOMA AND IMPLICATIONS FOR THERAPEUTIC STRATEGIES

Abstract INTRODUCTION Many glioblastoma (GBM) treatments fail because they treat tumor cells in isolation and ignore the surrounding microenvironment. In our phase 1b clinical trial of delivery of Topotecan (TPT) via Convection Enhanced Delivery (CED) in glioma, we demonstrated effective tumor cell elimination accompanied by a robust inflammatory response. We hypothesized that TPT induces an immunogenic response resulting in an unfavorable inflammatory landscape. METHODS To characterize chemotherapy’s effect on myeloid cells, we analyzed pre- and post-treatment biopsies from our trial using bulk RNA sequencing to measure canonical myeloid markers. We validated survival benefit of CED in vivo in our tumor model and characterized post-treatment tissue using single cell RNA sequencing (scRNA seq) after 7 days of treatment. We treated slice cultures generated from surgical samples of GBM with TPT and performed scRNA seq and histologic analysis to assess microenvironment changes after 24 hours of treatment. We used several in vitro assays to characterize the direct and indirect mechanisms of myeloid activation. RESULTS Bulk RNA sequencing of post treatment biopsies demonstrates upregulation of markers associated with activated and exhausted immune cell populations such as IBA1, CD68, MSR1, MARCO and loss of markers associated with homeostatic microglia such as P2RY12 and TMEM119. Similar results are found within 7 days of treatment in vivo, however, are not found in human derived samples treated acutely. Treatment of in vitro mono and co-culture systems demonstrate a tumor cell death dependent activation of myeloid cell populations via immunogenic molecules. CONCLUSIONS Chemotherapies eliminate proliferating glioma cells but also induce inflammation. While many investigators have tried to leverage this activation, our results suggest that chronic inflammation contributes to poor prognosis and recurrence. The identification of molecules responsible for this activation point to new therapeutic targets aimed at controlling treatment-induced inflammation, as part of a combinatorial approach to treat GBM.

IMMU-48. CRACKING THE ROLE OF ONCOMETABOLITES IN VIROIMMUNOTHERAPY FOR PEDIATRIC MALIGNANT BRAIN TUMORS

Abstract Diffuse midline gliomas (DMGs) are among the most frequent and lethal pediatric tumors. The standard of care for DMGs is palliative, resulting in a median survival of 12 months. Viroimmunotherapy is an emerging alternative treatment for these tumors. Our group developed a platform of oncolytic adenoviruses that was translated to the clinic. Specifically, Delta-24-RGD was recently tested in a Phase I clinical trial for patients with newly diagnosed DMG (NCT03178032) with encouraging results. To further stimulate the immune arm of this therapy, we developed Delta-24-RGDOX, expressing the T-cell activator OX40L (NCT03714334, NCT04714983). Using bulk RNAseq, we showed that treatment with Delta-24-RGDOX upregulated the immunosuppressive immunometabolic IDO-AhR pathway. Of importance, treatment with Th1-related cytokine IFNg resulted in the upregulation of IDO1 expression in a panel of human and murine DMG cells. In agreement with these data, we showed synergy between the oncolytic virus and several IDO1-AhR inhibitors in tumor and immune cell co-cultures, as indicated by T-cell activation and proliferation. To further dissect the reshaping of the TME in clinically relevant DMG models treated with Delta-24-RGD or Delta-24-RGDOX, we performed scRNA seq analysis. We showed a pronounced T-cell infiltration in virus-treated animals versus control-treated mice, further confirmed by flow cytometry. Additionally, Th1 and CD8 effector memory subsets were preferentially enriched in the Delta-24-RGDOX group, indicating an enhanced proinflammatory shift. Importantly, we detected, for the first time, upregulation of IDO-AhR downstream effectors in the myeloid compartment in the virus-treated groups. Interestingly, we detected IL4I1 in myeloid cells, a recently discovered checkpoint upstream of AhR never described in the context of virotherapy. Preliminary data using a combination of Delta-24-RGDOX and AhR inhibitors prolongs the survival of glioma-bearing mice. Collectively, our in vivo and in vitro data support the rationale to propel a future clinical trial combining Delta-24-RGDOX with AhR inhibitors for DMG.

EPCO-25. TRANSPOSABLE ELEMENTS INDUCE ONCO-EXAPTATION EVENTS IN MALIGNANT ATRX-DEFICIENT GLIOMAS

Abstract Deficiency of ATRX (Alpha Thalassemia/Mental Retardation Syndrome X-linked), a core member of SWI/SNF family chromatin regulator is altered in diffuse gliomas and coexist with IDH1/2 mutations in adults and histone G34R/V alterations in pediatric populations. Loss of ATRX has been associated with global alterations in chromatin accessibility and epigenomics landscape, however, the detail mechanism behind the oncogenic transformation remains elusive. Integrating transcriptomics and in-depth positional epigenome analysis over repetitive DNA sequences in progenitor and tumors models of murine and human gliomas, we describe ATRX-deficient gliomas is fundamentally a disease of altered Transposable Elements (TE’s) and regulates neuronal differentiation and cell motility in gliomas, as validated pharmacologically and genetically. Epigenome assessment reported de-repression of LINE-1 (Long interspersed nuclear elements-1), specifically at Lamina-Associated Domains (LADs) with gain of enhancer histone marks, with association in chromatin loops/topology in ATRX-deficient glioma cells. Moreover, our single cell-RNA seq analysis focusing on TE’s and molecular docking studies in human glioma stem cells demonstrated LINE-1 regulates oncogenic alternative splicing events and neuro-developmental signatures. Expanding the analysis, we identified a subset of evolutionary young regulatory TE’s to play critical role in ATRX-deficient gliomas involved in neuronal differentiation phenotype. To summarize, our cross-species analysis establishes tangible links between ATRX deficiency and multiscale dysregulated 3D-epigenomic architecture that hijack regulates onco-exaptation programs in gliomas. We further discover that enhancer-like TE’s fine-tune the transcriptional program with important clinical implications, that could help in developing treatment options in malignant gliomas.

DISP-17. OUTCOMES, CLINICAL AND MOLECULAR CHARACTERISTICS OF HISPANIC PATIENTS WITH IDH WILDTYPE GLIOBLASTOMA

Abstract BACKGROUND Knowledge about outcomes in Hispanic patients with gliomas is scant and mainly derived from tumor registries. IDH wild-type glioblastomas (GBM) are the most common and aggressive malignant primary brain tumor. Hispanics account for 19% of US population, reaching 29% in Florida and 65% in Miami-Dade County. As a reference center for GBM serving the community of South Florida we provide care for many Hispanic patients. Herein, we aim to describe the clinical and molecular features, including RNA seq analysis of Hispanic vs non-Hispanic patients with IDH wild-type GBM. METHODS Retrospective study of adult patients with diagnosis of IDH Wild-type Glioblastoma, who had NGS testing, treated at University of Miami. RESULTS 179 patients were included, 63 (35%) were Hispanic. 41 (65%) of Hispanic patients were males as compared to 65 (56%) of the non-Hispanics. Median age at diagnosis was 54 years (45-67) for the Hispanic vs 62 (54-72) years for non-Hispanic, p=.002. With a median follow up of 17 months (CI 95% 14-19), median overall survival for Hispanic patients was 22 months (18-25) and 23 months (18-28) for non-Hispanics, p=.99. 42 (23%) patients were enrolled in clinical trials, of whom 21 (50%) were Hispanic, meaning 33% of Hispanic patients enrolled. The molecular characteristics from these patients by next-generation sequencing, including RNA seq, are being analyzed. CONCLUSIONS In our series of IDH wild-type Glioblastomas, Hispanic patients were significantly younger at diagnosis compared to non-Hispanic patients, but there were no differences in overall survival. 33% of Hispanic patients were enrolled in clinical trials. As the latest growing ethnic group in the USA, major efforts have to be made to better understand the disease characteristics and outcomes of this particular population. To our knowledge this will be the first report of the molecular characteristics by NGS from Hispanic patients with GBM.

Multiomic Single Cell Profiling Identifies Drivers of Progression in T-Cell Prolymphocytic Leukemia

Treatments for T-cell prolymphocytic leukemia (T-PLL), while transiently effective in the frontline, have failed to achieve any substantial improvement in the outcomes of this ultra-rare chronic leukemia. Patients with relapsed T-PLL have a dismal prognosis, with a median overall survival (OS) of less than 6 months. For this study we used samples collected from: 8 patients with untreated T-PLL, 2 with relapsed/refractory (R/R) T-PLL, and 1 from a PDX model of R/R T-PLL. Whole-exome sequencing (WES) was used to elucidate the mutational landscape and single cell T-cell receptor sequencing (scTCRseq), coupled with single cell RNA sequencing (scRNA-seq), was used to generate gene expression and TCR clonality signatures at the single cell level. The WES revealed a higher frequency of mutations in R/R T-PLL compared to their baseline counterparts. R/R T-PLL samples displayed mutations of CXCR4 (37%; p.S312fs), STAT5B (46%; p.N642H), ATM (82%; p.L2890V), SAMHD1 (92%; p.A565T) and BCOR (p.N1585fs) and deletions in ASXL2 which were absent in treatment naïve T-PLL. In addition, all T-PLL samples harbored recurrent mutations on CXCR4, CD27, JAK1, STAT3, CCND1, CDKN1B, and the epigenetic regulators KMT2C, KMT2B, TET2, and KDM5C. The mutational profiles revealed the genomic complexity of T-PLL. The ubiquitous presence of activating mutations affecting the JAK/STAT axis underscored their importance in driving T-PLL survival. We also studied the diversity of TCR clonality and its relationship with pathogenesis and prognosis in T-PLL. All the samples exhibited dominance of at least one to up to 3 specific CDR3 sequences, revealing the anticipated clonal expansion in T-PLL. Expression of TCRα and TCRβ genes in the dominant clone differed among the samples.Monoclonality was validated as biallelic only when the 2nd largest transcripts of CDR3α or CDR3β clones were at least 5 times larger than its subceeder. The coupled TCR seq and RNA seq analysis displayed varied degrees of clonal expansions at a single cell resolution across all the patient samples. The T cell subsets were characterized by the expression of various T cell-markers across the cell clusters. In particular, 60% of the samples showed more expression of IL7R and lesser expression CD8A and CD8B, indicating their ability in suppressing the antitumor immunity. CD3E and CD3D were highly expressed across the clusters of T-PLL datasets.Out of 11 datasets, NKG7 and GZMA (cytotoxic T cell markers) were identified in more than 40% of the samples. The augmented levels of anti-apoptosis (BCL-2) and Jak/Stat signaling (JAK1, STAT3) markers were seen across the clusters of T-PLL scRNA seq datasets. The higher expression of PPP1R14B was found in 70% of the samples indicating its probable role in poor prognosis in T-PLL. In conclusion, T-PLL is characterized by numerous genomic aberrations, presence of 1-3 dominant clones, and gene expression signatures that facilitate survival, and resistance to apoptosis.

Human macrophages are immunnoprofiled by pericardial fluid small extracellular vesicles modulating lipid metabolism mechanisms

Abstract Background Coronary artery disease (CAD) is the main cause of myocardial infarction and consequent ischemic heart disease (IHD). Macrophages are central to CAD. Small extracellular vesicles (sEVs) can shuttle microRNAs and other molecular cargos from cell to cell, mediating response in target cells. Recent evidence supports the sEVs role in modulating macrophage phenotype. The pericardial fluid (PF) contains sEVs and immune cells including cavity-like macrophages. Purpose This study investigates whether PF-sEVs regulate macrophages, contributing to a specific immunophenotype in CAD patients. Methods PF was collected from CAD patients undergoing coronary bypass surgery (CABG) and non-atherosclerotic patients operated for mitral valve repair (control group). sEVs were isolated and characterised for size (Nanosight tracking analysi), tetrasapanin content (Nanoview chips) and microRNA content (RNA seq analysis). Monocytes from healthy donors were isolated from buffy coats and differentiated into macrophages following established protocols. Macrophages were incubated with either CAD-sEVs or non-CAD sEVs for 24h at 37oC. The cells were collected and processed for mRNA analyses (qRT-PCR) and flow cytometry. Further bioinformatics were employed to understand functional pathways targeted by PF-sEVs-miRNA. Results In line with the human-PF macrophages profile, exposure to CAD-sEVs reduces the anti-inflammatory profile of human macrophages. CAD-sEVs treated macrophages showed a CCR2-, CD36+low, CD206+low profile. While non-CAD-sEVs did not statistically differ from PBS nor untouched groups, CAD-sEVs increased the mRNA level of IL1a, IL1b, TNFa and decreased MRC1. Bioinformatic analysis showed that 861 miRNAs were decreased in the PF-sEVs from CAD patients compared to non-CAD. miRNA targets prediction and pathway analyses reported that clusters of PF-miRNAs could regulate CD36, decreased in PF-macrophages. Conclusions We demonstrate, that sEVs isolated from PF-CAD reduce the anti-inflammatory profile of human macrophages, and target crucial lipid metabolism pathways. These clinically relevant results could drive to decipher improved therapeutics to modulate the epicardial/myocardial immune response in CAD patients.

Antigen specificity of clonally-enriched CD8+ T cells in multiple sclerosis.

CD8+ T cells are the dominant lymphocyte population in multiple sclerosis (MS) lesions where they are highly clonally expanded. The clonal identity, function, and antigen specificity of CD8+ T cells in MS are not well understood. Here we report a comprehensive single-cell RNA-seq and T cell receptor (TCR)-seq analysis of the cerebrospinal fluid (CSF) and blood from a cohort of treatment-naïve MS patients and control participants. A small subset of highly expanded and activated CD8+ T cells were enriched in the CSF in MS that displayed high activation, cytotoxicity and tissue-homing transcriptional profiles. Using a combination of unbiased and targeted antigen discovery approaches, MS-derived CD8+ T cell clonotypes recognizing Epstein-Barr virus (EBV) antigens and multiple novel mimotopes were identified. These findings shed vital insight into the role of CD8+ T cells in MS and pave the way towards disease biomarkers and therapeutic targets.

7420 A Role for Glycoprotein Hormone Subunit Alpha 2 in Pituitary Development

Abstract Disclosure: L.T. Raetzman: None. X. Ge: None. K. Weis: None. Hypopituitarism results when the pituitary gland is not able to make one or more of its hormones. It is estimated that up to 80% of hypopituitarism detected at or near birth has no identifiable cause. We have created a mouse model of congenital hypopituitarism by conditionally deleting the gene Notch2 in the developing pituitary. NOTCH2 is expressed in pituitary stem cells and is necessary for stem cell maintenance, proliferation, and differentiation. However, the pathways NOTCH2 engages to affect pituitary development remain unclear. It is important to determine the mechanism of NOTCH2 action in the pituitary because it might help to reveal novel causes of hypopituitarism. In this study, we hypothesized that glycoprotein hormone subunit A2 (GPHA2), a corneal stem cell factor, is downstream of NOTCH2 signaling in the mouse pituitary. We found Gpha2 is expressed in a subset of quiescent Sox2-expressing pituitary stem cells by RNAscope in situ hybridization and scRNA seq analysis. A scRNA seq study in rats has also localized Gpha2 to Sox2-expressing folliculostellate cells. In Notch2 conditional knockout pituitaries, Gpha2 mRNA is reduced over 75% compared with control littermates. We then investigated the possible functions of GPHA2, hypothesizing it might have a role in stem cell maintenance and quiescence. However, pituitaries treated with a GPHA2 peptide do not have a change in proliferation or stem cell-related gene expression. Another role of GPHA2 may be as a paracrine signal to control lineage restricted cell fate. GPHA2 has been demonstrated to be a ligand for the thyroid stimulating hormone receptor (TSHR) and part of the hormone thyrostimulin. We demonstrated that, in dissociated adult pituitary cells, GPHA2 increased pCREB expression and this induction was reversed by co-treatment with a TSHR inhibitor. Our data show that TSHR is expressed in the rostral tip thyrotropes of the pars tuberalis and in isolated cells in the anterior lobe. These data suggest GPHA2 is a NOTCH2 related stem cell factor that activates TSHR signaling, potentially impacting pituitary development and maintenance as a paracrine signaling molecule. Presentation: 6/3/2024

Abstract B005: Characterizing the role of fibroadipogenic progenitors in pancreatic cancer cachexia: the role of miR-27a-3p

Abstract Introduction: Approximately 80% of patients with pancreatic cancer suffer from cachexia- a debilitating condition characterized by involuntary loss of muscle and fat. While muscle wasting has mostly been investigated from a whole tissue level, the impact of how different cell types contribute to muscle wasting remains elusive. The current study focuses on characterizing the role of muscle resident fibroadipogenic progenitors (FAPs) in pancreatic cancer cachexia. Dysregulation of FAPs produces sustained fibrogenic and/or adipogenic signals, which may lead to fibrofatty infiltration- a condition often observed in patients with cachexia. Using an experimental model of pancreatic cancer cachexia, we aim to understand (i) the overall contribution of FAPs to muscle wasting, (ii) the role of microRNAs (miRNAs- a class of small RNAs considered as global modulators of gene expression) in FAPs dysregulation and (iii) if modulating miRNAs in cancer improves muscle micro and macroenvironment. Methods: A male pancreatic cancer cell line was utilized to investigate the cachexia progression over time where day 14 (D14), 21 (D21), and day 26 (D26) were considered early, intermediate, and late time points, respectively. Mice were orthotopically implanted with 2 million pancreatic cancer cells (referred to as the KPC) and control mice received sham surgery (referred to as the control). Single nucleus RNA seq (snRNA seq) from quadriceps muscle, RNA seq, and miRNA profiling from sorted FAPs were performed. miR-27a-3p was knocked down in pancreatic cancer cells using a lentiviral system. Results: snRNA seq identified 13 clusters in quadriceps muscle including FAPs. Further, 4 subpopulations of FAPs with predominantly distinct transcriptional profiles were identified indicating that FAPs may be functionally heterogeneous. Further, muscle wasting genes such as Bmp4 and Osmr were present predominantly in FAPs suggesting that dysregulation of FAPs may contribute to muscle wasting. Time-resolved RNA seq analysis from sorted FAPs showed that alterations in adipogenesis (observed on day 14) preceded fibrosis (observed on d21), suggesting that dysregulation of FAPs is an early event in the muscle wasting trajectory, potentially contributing to fibrofatty infiltration. miR-27a-3p was chosen as a candidate molecule that was downregulated in FAPs and upregulated in the tumor when compared to controls. Inhibiting miR-27a-3p on FAPs increased adipogenesis and treatment with mimic reduced adipogenesis. Knocking down miR-27a in cancer prevented muscle wasting in vivo, and reduced FAPs dysregulation, suggesting that miR-27a-3p plays a dual role in tumor and muscle microenvironment. Conclusions: The study shows for the first time that dysregulation of FAPs significantly contributes to muscle wasting, potentially mediated through miR-27a-3p which when modulated in tumor reduces tumor progression and improves the muscle micro and macroenvironment. Citation Format: Ashok Narasimhan, Chun Wai Cheung, Nasim Kajabadi, Bruce Lin, Lin Wei Tung, Chihkai Chang, Fabio MV Rossi. Characterizing the role of fibroadipogenic progenitors in pancreatic cancer cachexia: the role of miR-27a-3p [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr B005.

Mast cells promote pathology and susceptibility in tuberculosis.

Tuberculosis (TB), caused by the bacterium Mycobacterium tuberculosis (Mtb), infects approximately one-fourth of the world's population. While most infected individuals are asymptomatic, latent TB infection (LTBI) can progress to cause pulmonary TB (PTB). We recently reported an increased accumulation of mast cells (MCs) in lungs of macaques with PTB, compared with LTBI in macaques. MCs respond in vitro to Mtb exposure via degranulation and by inducing proinflammatory cytokines. In the current study, we show the dominant production of chymase by MCs in granulomas of humans and macaques with PTB. Using scRNA seq analysis, we show that MCs found in LTBI and healthy lungs in macaques are enriched in genes involved in tumor necrosis factor alpha, cholesterol and transforming growth factor beta signaling. In contrast, MCs clusters found in PTB express transcriptional signatures associated with interferon gamma, oxidative phosphorylation, and MYC signaling. Additionally, MC deficiency in the mouse model showed improved control of Mtb infection that coincided with reduced accumulation of lung myeloid cells and diminished inflammation at chronic stages. Thus, these collective results provide novel evidence for the pathological contribution of MCs during Mtb infection and may represent a novel target for host directive therapy for TB.

Bulk-RNA and single-nuclei RNA seq analyses reveal the role of lactate metabolism-related genes in Alzheimer's disease.

Dysfunctional lactate metabolism in the brain has been implicated in neuroinflammation, Aβ deposition, and cell disturbance, all of which play a significant role in the pathogenesis of Alzheimer's disease (AD). In this study, we aimed to investigate the lactate metabolism-related genes (LMRGs) in AD via an integrated bulk RNA and single-nuclei RNA sequencing (snRNA-seq) analysis, with a specific focus on microglia. We obtained 26 HC and 24 AD snRNA-seq samples originated from human prefrontal cortex in Gene Expression Omnibus (GEO) database and collected 873 LMRGs from three databases, namely MSigDB, The Human Protein Atlas and GeneCards. Bulk RNA was analyzed with LMRG characteristics in AD by using Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), the protein-protein interaction (PPI), CytoHubba-MCC, Support Vector Machine (SVM) algorithms analyses. Then we conducted the Receiver Operating Characteristic (ROC) curve, correlation, and connection network analyses for biomarkers. Their differential expression validation was performed using AlzData database. The single-nuclei RNA analysis of microglia was applied to identify hub genes and pathways using cell-cell communication analysis and high dimensional Weighted Gene Co-Expression Network Analysis (hdWGCNA). Support Vector Machine (SVM) algorithm showed an AUC of 0.967, a sensitivity of 93.30% and a specificity of 100.00%. Our analysis identified biomarkers with LMRG characteristics, namely INSR, CDKL1, and PNISR. ROC analysis revealed that each of these biomarkers exhibited excellent diagnostic potential, as evidenced by their respective area under the curve (AUC) values: INSR (AUC: 0.679), CDKL1 (AUC: 0.788), and PNISR (AUC: 0.724). Correlation analysis showed that biomarkers exhibited a positive correlation with each other. Connection network illustrated their shared biological processes: aging, phosphorylation, metabolic process, and apoptosis. Cell-cell communication analysis revealed that GALECTIN signaling pathway was exclusively expressed in AD microglia, and only LGALS9 exhibited significant overexpression. HdWGCNA identified FTH1 as a hub gene enriched in ferroptosis and mineral absorption pathways within microglia. The roles of INSR, CDKL1, PNISR, LGALS9, and FTH1 should be taken into account to enhance our understanding of lactate metabolism in the context of AD.

Mesenchymal stromal/stem cell tissue source and in vitro expansion impact extracellular vesicle protein and miRNA compositions as well as angiogenic and immunomodulatory capacities.

Recently, therapies utilizing extracellular vesicles (EVs) derived from mesenchymal stromal/stem cells (MSCs) have begun to show promise in clinical trials. However, EV therapeutic potential varies with MSC tissue source and in vitro expansion through passaging. To find the optimal MSC source for clinically translatable EV-derived therapies, this study aims to compare the angiogenic and immunomodulatory potentials and the protein and miRNA cargo compositions of EVs isolated from the two most common clinical sources of adult MSCs, bone marrow and adipose tissue, across different passage numbers. Primary bone marrow-derived MSCs (BMSCs) and adipose-derived MSCs (ASCs) were isolated from adult female Lewis rats and expanded in vitro to the indicated passage numbers (P2, P4, and P8). EVs were isolated from the culture medium of P2, P4, and P8 BMSCs and ASCs and characterized for EV size, number, surface markers, protein content, and morphology. EVs isolated from different tissue sources showed different EV yields per cell, EV sizes, and protein yield per EV. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses of proteomics data and miRNA seq data identified key proteins and pathways associated with differences between BMSC-EVs and ASC-EVs, as well as differences due to passage number. In vitro tube formation assays employing human umbilical vein endothelial cells suggested that both tissue source and passage number had significant effects on the angiogenic capacity of EVs. With or without lipopolysaccharide (LPS) stimulation, EVs more significantly impacted expression of M2-macrophage genes (IL-10, Arg1, TGFβ) than M1-macrophage genes (IL-6, NOS2, TNFα). By correlating the proteomics analyses with the miRNA seq analysis and differences observed in our in vitro immunomodulatory, angiogenic, and proliferation assays, this study highlights the trade-offs that may be necessary in selecting the optimal MSC source for development of clinical EV therapies.

Novel miRNA-inducing drugs enable differentiation of retinoic acid-resistant neuroblastoma cells.

Tumor cell heterogeneity in neuroblastoma, a pediatric cancer arising from neural crest-derived progenitor cells, poses a significant clinical challenge. In particular, unlike adrenergic (ADRN) neuroblastoma cells, mesenchymal (MES) cells are resistant to chemotherapy and retinoid therapy and thereby significantly contribute to relapses and treatment failures. Previous research suggested that overexpression or activation of miR-124, a neurogenic microRNA with tumor suppressor activity, can induce the differentiation of retinoic acid-resistant neuroblastoma cells. Leveraging our established screen for miRNA-modulatory small molecules, we validated PP121, a dual inhibitor of tyrosine and phosphoinositide kinases, as a robust inducer of miR-124. A combination of PP121 and BDNF-activating bufalin synergistically arrests proliferation, induces differentiation, and maintains the differentiated state of MES SK-N-AS cells for 8 weeks. RNA-seq and deconvolution analyses revealed a collapse of the ADRN core regulatory circuitry (CRC) and the emergence of novel CRCs associated with chromaffin cells and Schwann cell precursors. Using a similar protocol, we differentiated and maintained MES neuroblastoma GI-ME-N and SH-EP cell lines, as well as glioblastoma LN-229 and U-251 cell lines, for over 16 weeks. In conclusion, our novel protocol suggests a promising treatment for therapy-resistant cancers of the nervous system. Moreover, these long-lived, differentiated cells provide valuable models for studying mechanisms underlying differentiation, maturation, and senescence.